Rybp interacts with Hippi and enhances Hippi-mediated apoptosis

Rybp (DEDAF) has been shown to interact with DED-containing proteins and to encode pro-apoptotic functions. Here we characterize a novel interaction between Rybp and Hippi, a protein implicated in neuronal apoptosis as well as in the pathogenesis of Huntington’s disease. Rybp can synergize with Hippi to enhance Caspase 8-mediated apoptosis and also appears to be essential for Hippi-mediated apoptosis. Moreover, Rybp may mediate or regulate the interaction between Hippi and Caspase 8. Finally, Rybp and Hippi co-localize in a subset of neurons in the developing mouse brain. Together, these findings suggest that Rybp and Hippi may functionally interact in the apoptotic processes that accompany normal murine neural development.     

Reduced glutathione levels affect the culmination and cell fate decision in Dictyostelium discoideum

Glutaredoxins have been known to be glutathione-dependent oxidoreductases that participate in the redox regulation of various cellular processes. To understand the role of glutaredoxins in the development, we examined glutaredoxin 1 (Grx1) of Dictyostelium discoideum. Its mRNA was highly accumulated at the mound and the culmination stages. When Grx1-overexpressing cells were developed, their culmination was delayed, and the expression of marker genes for prespore and spore decreased. Interestingly, they had about 1.5-fold higher amount of reduced glutathione (GSH) compared with parental cells and their prolonged migration was repressed by the oxidant such as hydrogen peroxide. To confirm the effect of GSH on the culmination, glutathione reductase (Gsr) was overexpressed or underexpressed. Similar to Grx1-overexpressing cells, Gsr-overexpressing cells contained about 1.5-fold higher amount of GSH and exhibited the delayed culmination. In contrast, the knockdown mutant of Gsr had nearly 50% lower amount of GSH and showed accelerated culmination. Taken together, these data suggest that the culmination of Dictyostelium is controlled by GSH. In addition, the cells having higher GSH levels showed a prestalk tendency in the chimeric slugs with parental cells, indicating that the difference in the amount of GSH may affect the determination of cell fate.     

Apoptin: Specific killer of tumor cells?

In the early 1990s it was discovered that the VP3/Apoptin protein encoded by the Chicken Anemia virus (CAV) possesses an inherent ability to specifically kill cancer cells. Apoptin was found to be located in the cytoplasm of normal cells while in tumor cells it was localized mainly in the nucleus.¹ These differences in the localization pattern were suggested to be the main mechanism by which normal cells show resistance to Apoptin-mediated cell killing. Although the mechanism of action of Apoptin is presently unknown, it seems to function by the induction of programmed cell death (PCD) after translocation from the cytoplasm to the nucleus and arresting the cell cycle at G₂/M, possibly by interfering with the cyclosome.² In addition, cancer specific phosphorylation of Threonine residue 108 has been suggested to be important for Apoptin’s function to kill tumor cells.³ In contrast to the large number of publications reporting that nuclear localization, induction of PCD and phosphorylation of Apoptin is restricted to cancer cells, several recent studies have shown that Apoptin has the ability to migrate to the nucleus and induce PCD in some of the normal cell lines tested. There is evidence that high protein expression levels as well as the cellular growth rate may influence Apoptin’s ability to specifically kill tumor cells. Thus far both in vitro and in vivo studies indicate that Apoptin is a powerful apoptosis inducing protein with a promising prospective utility in cancer therapy. However, here we show that several recent findings contradict some of the earlier results on the tumor specificity of Apoptin, thus creating some controversy in the field. The aim of this article is to review the available data, some published and some unpublished, which either agree or contradict the reported “black and white” tumor cell specificity of Apoptin. Understanding what factors appear to influence its function should help to develop Apoptin into a potent anti-cancer agent.     

The viral death protein Apoptin interacts with Hippi,the protein interactor of Huntingtin-interacting protein 1

Apoptin, a chicken anemia virus-encoded protein, induces apoptosis in human tumor cells but not in normal cells. The tumor-specific activity of Apoptin is correlated with its nuclear localization in tumor cells. In an attempt to elucidate the molecular mechanism of Apoptin-induced apoptosis, we identified human Hippi, the protein interactor and apoptosis co-mediator of Huntingtin interacting protein 1, as one of the Apoptin-associated proteins by yeast two-hybrid screen. We also demonstrated that Hippi could interact with Apoptin both in vitro and in human cells. Furthermore, subcellular localization studies showed that Hippi and Apoptin perfectly colocalized in the cytoplasm of normal human HEL cells, whereas in cancerous HeLa cells most Apoptin and Hippi were located separately in the nucleus and cytoplasm and, thus, showed only a modest colocalization. Mapping studies indicate that Hippi binds within the self-multimerization domain of Apoptin, and Apoptin binds to the C-terminal half of Hippi, including its death effector domain-like motif. Our results suggest that the Apoptin-Hippi interaction may play a role in the suppression of apoptosis in normal cells.     

Huntingtin-interacting protein 1-mediated neuronal cell death occurs through intrinsic apoptotic pathways and mitochondrial alterations

Huntingtin interacting protein-1 (Hip1) is known to be associated with the N-terminal domain of huntingtin. Although Hip1 can induce apoptosis, the exact upstream signal transduction pathways have not been clarified yet. In the present study, we examined whether activation of intrinsic and/or extrinsic apoptotic pathways occurs during Hip1-mediated neuronal cell death. Overexpression of Hip1 induced cell death through caspase-3 activation in immortalized hippocampal neuroprogenitor cells. Interestingly, proteolytic processing of Hip1 into partial fragments was observed in response to Hip1 transfection and apoptosis-inducing drugs. Moreover, Hip1 was found to directly bind to and activate caspase-9. This promoted cytosolic release of cytochrome c and apoptosis-inducing factor via mitochondrial membrane perturbation. Furthermore, Hip1 could directly bind to Apaf-1, suggesting that the neurotoxic signals of Hip1 transmit through the intrinsic mitochondrial apoptotic pathways and the formation of apoptosome complex.     

The viral death effector Apoptin reveals tumor-specific processes

Several natural proteins, including the cellular protein TRAIL and the viral proteins E4orf4 and Apoptin, have been found to exert a tumor-preferential apoptotic activity. These molecules are potential anti-cancer agents with direct clinical applications. Also very intriguing is their possible utility as sensors of the tumorigenic phenotype. Here, we focus on Apoptin, discussing recent research that has greatly increased our understanding of its tumor-specific processes. Apoptin, which kills tumor cells in a p53- and Bcl-2-independent, caspase-dependent manner, is biologically active as a highly stable, multimeric complex consisting of 30 to 40 monomers that form distinct superstructures upon binding cooperatively to DNA. In tumor cells, Apoptin is imported into the nucleus prior to the induction of apoptosis; this contrasts with the situation in primary or low-passage normal cell cultures where nuclear translocation of Apoptin is rare and inefficient. Apoptin contains two autonomous death-inducing domains, both of which exhibit a strong correlation between nuclear localization and killing activity. Nevertheless, forced nuclear localization of Apoptin in normal cells is insufficient to allow induction of apoptosis, indicating that another activation step particular to the tumor or transformed state is required. Indeed, a kinase activity present in cancer cells but negligible in normal cells was recently found to regulate the activity of Apoptin by phosphorylation. However, in normal cells, Apoptin can be activated by transient transforming signals conferred by ectopically expressed SV40 LT antigen, which rapidly induces Apoptins phosphorylation, nuclear accumulation and the ability to induce apoptosis. The region on LT responsible for conferring this effect has been mapped to the N-terminal J domain. In normal cells that do not receive such signals, Apoptin becomes aggregated, epitope-shielded and is eventually degraded in the cytoplasm. Finally, Apoptin interacts with various partners of the human proteome including DEDAF, Nmi and Hippi, which may help to regulate either Apoptins activation or execution processes. Taken together, these recent advances illustrate that elucidating the mechanism of Apoptin-induced apoptosis can lead to the discovery of novel tumor-specific pathways that may be exploitable as anti-cancer drug targets.