Masayuki Kanai

Loss of poly(ADP-ribose) glycohydrolase causes progressive neurodegeneration in Drosophila melanogaster

Poly(ADP-ribosyl)ation has been suggested to be involved in regulation of DNA repair, transcription, centrosome duplication, and chromosome stability. However, the regulation of degradation of poly(ADP-ribose) and its significance are not well understood. Here we report a loss-of-function mutant Drosophila with regard to poly(ADP-ribose) glycohydrolase, a major hydrolyzing enzyme of poly(ADP-ribose). The mutant lacks the conserved catalytic domain of poly(ADP-ribose) glycohydrolase, and exhibits lethality in the larval stages at the normal development temperature of 25°C. However, one-fourth of the mutants progress to the adult stage at 29°C but showed progressive neurodegeneration with reduced locomotor activity and a short lifespan. In association with this, extensive accumulation of poly(ADP-ribose) could be detected in the central nervous system. These results suggest that poly(ADP-ribose) metabolism is required for maintenance of the normal function of neuronal cells. The phenotypes observed in the parg mutant might be useful to understand neurodegenerative conditions such as the Alzheimers and Parkinsons diseases that are caused by abnormal accumulation of substances in nervous tissue.

Involvement of poly(ADP-Ribose) polymerase 1 and poly(ADP-Ribosyl)ation in regulation of centrosome function.

ABSTRACT The regulatory mechanism of centrosome function is crucial to the accurate transmission of chromosomes to the daughter cells in mitosis. Recent findings on the posttranslational modifications of many centrosomal proteins led us to speculate that these modifications might be involved in centrosome behavior. Poly(ADP-ribose) polymerase 1 (PARP-1) catalyzes poly(ADP-ribosyl)ation to various proteins. We show here that PARP-1 localizes to centrosomes and catalyzes poly(ADP-ribosyl)ation of centrosomal proteins. Moreover, centrosome hyperamplification is frequently observed with PARP inhibitor, as well as in PARP-1-null cells. Thus, it is possible that chromosomal instability known in PARP-1-null cells can be attributed to the centrosomal dysfunction. P53 tumor suppressor protein has been also shown to be localized at centrosomes and to be involved in the regulation of centrosome duplication and monitoring of the chromosomal stability. We found that centrosomal p53 is poly(ADP-ribosyl)ated in vivo and centrosomal PARP-1 directly catalyzes poly(ADP-ribosyl)ation of p53 in vitro. These results indicate that PARP-1 and PARP-1-mediated poly(ADP-ribosyl)ation of centrosomal proteins are involved in the regulation of centrosome function.

Interaction between ROCK II and Nucleophosmin/B23 in the Regulation of Centrosome Duplication

ABSTRACT Nucleophosmin (NPM)/B23 has been implicated in the regulation of centrosome duplication. NPM/B23 localizes between two centrioles in the unduplicated centrosome. Upon phosphorylation on Thr199 by cyclin-dependent kinase 2 (CDK2)/cyclin E, the majority of centrosomal NPM/B23 dissociates from centrosomes, but some NPM/B23 phosphorylated on Thr199 remains at centrosomes. It has been shown that Thr199 phosphorylation of NPM/B23 is critical for the physical separation of the paired centrioles, an initial event of the centrosome duplication process. Here, we identified ROCK II kinase, an effector of Rho small GTPase, as a protein that localizes to centrosomes and physically interacts with NPM/B23. Expression of the constitutively active form of ROCK II promotes centrosome duplication, while down-regulation of ROCK II expression results in the suppression of centrosome duplication, especially delaying the initiation of centrosome duplication during the cell cycle. Moreover, ROCK II regulates centrosome duplication in its kinase and centrosome localization activity-dependent manner. We further found that ROCK II kinase activity is significantly enhanced by binding to NPM/B23 and that NPM/B23 acquires a higher binding affinity to ROCK II upon phosphorylation on Thr199. Moreover, physical interaction between ROCK II and NPM/B23 in vivo occurs in association with CDK2/cyclin E activation and the emergence of Thr199-phosphorylated NPM/B23. All these findings point to ROCK II as the effector of the CDK2/cyclin E-NPM/B23 pathway in the regulation of centrosome duplication.

Subcellular localization of poly(ADP-ribose) glycohydrolase in mammalian cells.

Posttranslational modification plays important roles in a range of cellular functions. Poly(ADP-ribosyl)ation influences DNA repair, transcription, centrosome duplication, and chromosome stability. Poly(ADP-ribose) attached to acceptor proteins should be properly hydrolyzed by poly(ADP-ribose) glycohydrolase (PARG). However the subcellular localization and the role of PARG have not been well characterized. Here, we transiently expressed GFP- or Myc-tagged human PARG in mammalian cells and revealed that the subcellular distribution of human PARG changes dramatically during the cell cycle. GFP-hPARG is found almost exclusively in the nucleus during interphase. During mitosis, most GFP-hPARG protein localizes to the cytoplasm and hardly any GFP-hPARG protein is found associated with the chromosomes. Furthermore, we found that GFP-hPARG localizes to the centrosomes during mitosis. Our findings suggest that shuttling of PARG between nucleus and cytoplasm and proper control of poly(ADP-ribose) metabolism throughout the cell cycle may play an important role in regulating cell cycle progression and centrosome duplication.

Electrochemical analysis of single nucleotide polymorphisms of p53 gene.

Single nucleotide polymorphisms (SNPs) of cancer repression gene p53 were analyzed electrochemically with ferrocenyl naphthalene diimide (1) as a hybridization indicator. The SNPs studied were the transition to A from G in the codon for amino acid at positions 175, 248 or 273 and the transversion to C from G in the codon for the amino acid at position 72. Thus, 20-meric oligonucleotides carrying the SNP site were used both as a sample and a probe with the latter immobilized on an electrode. Even one base difference on the p53 gene resulted in a significant difference in the current response of 1 and the magnitude of the response correlated with the amount of the DNA hybrid on the electrode. Moreover, when PCR products of exon 4, on which the P72/R72 SNP resides, of the p53 gene were analyzed by this method, the heterozygote and homozygotes were discriminated with modest precision.

Enhanced expression of MYCN leads to centrosome hyperamplification after DNA damage in neuroblastoma cells

Centrosomes play important roles in cell polarity, regulation of cell cycle and chromosomal stability. Centrosome abnormality is frequently found in many cancers and contributes to chromosomal instability (including aneuploidy, tetraploidy, and/or micronuclei) in daughter cells through the assembly of multipolar or monopolar spindles during mitosis. It has recently been reported that loss of tumor suppressor genes or overexpression of oncogenes causes centrosome hyperamplification. Amplification and overexpression of the MYCN oncogene is found in a subgroup of neuroblastomas. In this study, we examined whether overexpression of MYCN causes centrosome hyperamplification in neuroblastoma cells. We show that ectopic expression of MYCN alone in a neuroblastoma cell line did not cause centrosome hyperamplification. However, centrosome hyperamplification and micronuclei formation were seen in these cells after DNA damage. These findings suggest that overexpression of MYCN abrogates the regulation of the centrosome cycle after DNA damage.

Roles of Poly(ADP-Ribose) Metabolism in the Regulation of Centrosome Duplication and in the Maintenance of Neuronal Integrity

The chemical structure of poly(ADP-ribose) suggests not only that its modification of acceptor proteins should modify the structure and function of the acceptor proteins, but also that the poly(ADP-ribose) molecule itself should possess an intrinsic structural information that can alter cellular function(s).