Asy (apoptosis/saibousi Yutsudo) is a novel apoptosis-inducing gene found in 1999 by Yutsudo group in Japan. In 2000, Qi Bing et al. cloned another novel gene, named hap (homologue of ASY protein), which encoded the ASY interacting protein, from human lung cell line (WI-38) cDNA library by using yeast two-hybrid system. It has been proved that ASY formed homodimer in yeast and human cell line, ASY and HAP formed heterodimer in yeast cells, and both induced cell apoptosis in human tumor cell lines Sao2 and CGL4. This paper showed that HAP could form homodimer in yeast cells by yeast two-hybrid system; HAP and ASY could produce heterodimer in human cell line by cross-immunoprecipitation test; by using apoptosis-testing technologies such as AnnexinV, TUNEL, DNA ladder and Flow Cytometry, the cell apoptosis in human normal or tumor cell lines transfected with hap or asy individually or cotransfected by the both was qualified or quantified. It was firstly demonstrated that ASY or HAP induced cell apoptosis not only in human tumor cell lines, but also in human normal cell lines. Moreover, we proved that the heterodimer between ASY and HAP decreased apoptosis-inducing activity from the homodimer of ASY or HAP. It revealed that by choosing to form heterodimer or homodimer between ASY and/or HAP is an important mechanism of regulating apoptosis in human cell lines.

In attempting to produce the HAP, endoplasmic reticulum (ER) targeted apoptosis-inducing protein, as a GSTfusion protein we found that the expression of HAP, but not GST alone, induced bacterial cell death. The HAP protein inhibited the bacterial growth within 30min after inducting HAP expression. The transmission electron microscopic examination revealed that the morphology of the bacterial cells expressing hap was changed dramatically: unusually elongated phenotype compared with those of controls and finally leading to cell death. The lethality of HAP was relieved by the addition of vitamin E as a reducing agent and under anaerobic growth conditions. These results suggest that a trace amount of HAP induces bacterial cell death and the death is related with reactive oxygen species (ROS).

Although apoptosis plays an essential role in the embryogenesis and homeostasis of multicellular organ-isms, this mechanism has not yet been fully clarified. We isolated a novel human apoptosis-inducing gene, ASY, which encodes an endoplasmic reticulum-targeting pro-tein without any known apoptosis-related motifs. This gene is identical to the Nogo-B, a splice variant of the Nogo-A which has recently been shown to be an inhibitor of neuronal regeneration in the central nervous system. Ectopic expression of the ASY gene led to extensive apoptosis, particularly in cancer cells. Furthermore, transcription of the ASY gene was suppressed in small cell lung cancer. These results suggest that a new type of apoptosis-inducing gene, namely, ASY, may be involved in the development of certain types of cancer. Oncogene (2001) 20, 3929-3936.

We have previously shown that ectopic expression of the ASY/Nogo-B gene induced apoptosis in various cancer cell lines. Nogo-A, a splice variant of the ASY, has been reported to have an inhibitory effect on neuronal regeneration in the central nervous system. To investigate the mechanism of ASY-induced apoptosis or inhibition of neuronal regeneration, we cloned a cDNA for the ASY-interacting protein from the human cDNA library using the yeast two-hybrid method, and obtained a cDNA we designated as ASYIP. The ASYIP protein contains two hydrophobic regions and a double lysine endoplasmic reticulum (ER) retrieval motif at its C-terminus, which was shown to be identical to RTN3, a reticulon family protein of unknown function. We showed that ASY and ASYIP proteins formed a complex also in human cells. Mutational analysis indicated that both of the hydrophobic regions of the ASYIP protein were required for the association. By immunofluorescence analysis, the ASYIP protein was shown to be co-localized with ASY in the ER. Characterization of the ASYIP gene may be very useful in clarifying the mechanism of ASY-induced apoptosis or Nogo-involved inhibition of neuronal regeneration in the central nervous system. J. Cell. Physiol. 196: 312-318, 2003.

asy gene is a novel apoptosis-inducing gene, but its mechanism is unclear. To investigate the mechanism of asy inducing apoptosis, a novel gene encoding ASY interacting protein (asyip) is isolated from human lung cell line (WI-38) cDNA library with yeast two-hybrid system. The asyip gene is constitutively expressed as two mRNA transcripts with the size of 1.8 and 2.7kb in various human tissues at different levels. Sequence analysis of full-length cDNA reveals that the two alternative transcripts of asyip gene contain common 5' end and different 3' end, and share a common open reading frame encoding a polypeptide of 236 amino acids. Two protein kinase C phosphorylation sites and two casein kinase II phosphorylation sites are found in ASYIP amino acid sequence. Two highly hydrophobic regions encoding potentially two transmembrane domains are present. The ASYIP protein contains a C-terminal endoplasmic reticulum retrieval signal (Lys-Lys-Lys-Ala-Glu). Immunoprecipitation assay confirmed the interaction of ASY and ASYIP in mammalian cells. Compared with asy gene, overexpression of asyip gene can inhibit growth of tumor cell Saos2 and induce cell apoptosis with a low efficiency.

Two antiapoptotic types of genes, iap and p35, were found in baculoviruses. P35 is a 35-kDa protein that can suppress apoptosis induced by virus infection or by diverse stimuli in vertebrates or invertebrates. iap homologues were identified in insects and mammals. Recently, we have identified sl-p49, a novel apoptosis suppressor gene and the first homologue of p35, in the genome of the Spodoptera littoralis nucleopolyhedrovirus. Here we show that sl-p49 encodes a 49-kDa protein, confirmed its primary structure that displays 48.8% identity to P35, and performed computer-assisted modeling of P49 based on the structure of P35. We demonstrated that P49 is able to inhibit insect and human effector caspases, which requires P49 cleavage at Asp94. Finally we identified domains important for P49's antiapoptotic function that include a reactive site loop (RSL) protruding from a B-barrel domain. RSL begins at an amphipathic a1 helix, traverses the a-sheet central region, exposing Asp94 at the apex, and rejoins the a-barrel. Our model predicted seven a-helical motifs, three of them unique to P49. a-Helical motifs a1, a2, and a4a were required for P49 function. The high structural homology between P49 and P35 suggests that these molecules bear a scaffold common to baculovirus "apoptotic suppressor" proteins. P49 may serve as a novel tool to analyze the contribution of different components of the caspase chain in the apoptotic response in organisms not related

Baculoviruses possess two types of genes that suppressed apoptosis, p35 and inhibitor of apoptosis (iap). In this study we report the isolation and identification of an inhibitor of apoptosis gene Sliap in the genome of the Spodoptera littoralis nucleopolyhedrvirus (SINPV). The Sliap sequence predicted a 15kDa polypeptide with only on BIR domain and a RING finger, both motifs characteristic of the IAP family of proteins, and a third specific acidic-rich motif. These characteristics, shared with the Spodoptera littura NPV IAP2/3, Epiphyas postvittana NPV IAP4, Lymantria dispar NPV IAP and Orgyia pseudotsugata NPV IAP4 (Orf 107) allowed us to classify them in a new homology group (IAP-4). Sliap was able to delay, but not to suppress,apoptosis induced by replication of a recombinant AcMNPV deficient in p35. In SINPV infected-SF9 cells Sliap was expressed earlier than sl-p49 suggesting that its role at the initiation of infection was to delay the apoptotic response of the host.

A novel cloned Spodoptera littoralis Nucleopolyhedrovirus (SlNPV) p49 gene is able to suppress apoptosis of insect cells Sf9 triggered by virus. The amino acid sequence of P49 expressed in baculovirus expression system is the same as predicted, indicating that the expression of P49 is correct. Metabolic labeling revealed that p49 was able to be expressed both in the early and late phases after the viral infection, and only in the late phase was the expression driven by polyhedra promoter, but the amount of expression was higher than that of wtSlNPV. In summary, the early gene of SlNPV p49 as well as p35 of AcMNPV is able to be expressed in the late phase, but its promoter is weaker compared with polyhedra promoter. In vitro, P49 can be cut by Bm caspase and human caspase-3, yielding 10 and 40 ku fragments. Purified P49 blocks the substrate cleavage by Bm caspase and human caspase-3, showing that P49 inhibits downstream caspases in the apoptotic pathway.

An antitumour lectin (named AAL) consisting of two identical subunits of 15.8 kDa was isolated from the fruiting bodies of the edible mushroom Agrocybe aegerita using a procedure which involved precipitating the extract by addition of (NH4)2SO4, ion exchange chromatography on DEAE-Sepharose Fast Flow, gel filtration chromatography on Sephacryl S-200 HR and finally purification on a GF-250 HPLC column. Amino acid analysis of the N-terminus and an internal fragment indicated that the sequences of the two fragments were QGVNIYNI and Q(K)-PDGPWLVEK(Q)R respectively. AAL showed strong inhibition of the growth of human tumour cell lines HeLa, SW480, SGC-7901, MGC80-3, BGC-823, HL-60 and mouse sarcoma S-180. AAL also inhibited the viability of S-180 tumour cells in vivo.Analysis by Hoechst 33258 staining, MitoSensor Kit and flow cytometry showed that AAL induced apoptosis in HeLa cells. TUNEL (terminal transferase deoxytidyl uridine end labelling) analysis of slides of tumour tissues excised from BALB/c mice also demonstrated the apoptosis-induction activity of the lectin. Furthermore, AAL was shown to possess DNase activity in assays using plasmid pCDNA3 and salmon sperm DNA. Based on the results obtained in these assays, we conclude that AAL exerts its antitumour effects via apoptosis-inducing and DNase activities.

Apoptosis serves as an important defense strategy employed by host cells against viral invasion. Many viruses contain the antiapoptotic genes to block the defense-by-death response of host cells. In this study, we tried to identify the putative anti-apoptotic genes in white spot syndrome virus (WSSV) genome. We confirmed that actinomycin D could induce apoptosis of shrimp primary cells. However, the apoptosis triggered by actinomycin D was inhibited by WSSV infection. As mutants of Autographa californica nucleopolyhedrovirus (AcMNPV), AcMNPVD35k/pol+ lacks a functional P35 gene undergoing apoptosis and its infection could induce Sf9 cell apoptosis. To identify the putative apoptotic suppressor gene of WSSV, overlapping cosmid clones representing the entire WSSV genome were individually cotransfected along with genome DNA of AcMNPVDP35k/pol+. Using this marker rescue assay, a WSSV DNA fragment that was able to rescue AcMNPVDP35k/pol+ infection in Sf9 cells was isolated. By further sequence analysis and rescue assay, the ORF390 was identified as a novel anti-apoptotic gene. The ORF displays two putative caspase9 cleavage sites LLVETDGPS, VKLEHDGSK, and a caspase3 cleavage site EEDEVDGVP. The ORF was cloned into the pIE1 vector and then the recombinant vector was transfected into Sf9 cells. The Sf9 cells did not show obvious characteristics of apoptosis when infected with AcMNPVDP35k/pol+. And the transient expression of ORF390 allowed AcMNPVDP35k/pol+ replication in Sf9 cells and resulted in the formation of polyhedra successfully. The results indicate that function of ORF390 in WSSV is a kind of apoptotic suppressor like P35 in AcMNPV.