The Apoptotic Pathways And Intracellular Function Of Two Novel ER-localized Proteins

Reticulon (RTN) family is a relatively new eukaryotic gene family with unknown functions but broad expression and peculiar topological features. Relatively recently Reticulon (RTN) was described to code a family of proteins in higher vertebrates, which were characterized by two homologous large hydrophobic transmembrane domains and a potential C-terminal endoplasmic reticulum retrieval signal. At least 4 different RTN genes (RTN1, -2, -3, and -4/Nogo) have been identified in mammals, and over 250 reticulon-like (RTNL) genes were identified in deeply diverging eukaryotes, fungi, plants, and animals. The overall occurrence of RTNs in different phyla and the evolution of the RTN gene family emphasized on the one hand the universal role of reticulons in the eukaryotic system and on the other hand the acquisition of putative new functions through acquirement of novel amino-terminal exons. Except for RTN4-A/Nogo-A, thought to be an inhibitor for neurite outgrowth, restricting the regenerative capabilities of the mammalian CNS after injury and RTN4-B/Nogo-B/ASY, thought to be a pro-apoptotic protein, several newest discoveries showed that the reticulon were involved in vesicle trafficking events including regulated exocytosis and endocytosis, a regulator of vascular remodeling, a negative modulators of BACE1 and the production of amyloid-beta.ASY/Nogo-B was recently isolated as an apoptosis-inducing gene that encodes an ER-targeting protein and is downregulated in small cell lung cancer. We have identified and characterized a novel ASY interaction protein, designated as HAP(homologue of ASY rjrotein, also named AS YIP, ASY interacting protein, which was also localized at ER. Analysis of protein demonstrated that HAP was a reticulon (RTN) family protein RTN3/HAP, and ASY was RTN4B/Nogo-B, characterized by two transmembrane domains and a potential C-terminal endoplasmic reticulum retrieval signal. Our previous work demonstrated that the overexpression of RTN3/HAP caused apoptosis with the depletion of endoplasmic reticulum (ER) Ca2+ stores and the elevation of cytosolic Ca2+. However, we know little about the pro-apoptotic mechanism and function of RTN3/HAP and ASY.In present study, we revealed that RTN3/HAP protein induced endoplasmic reticulum overload response, mediated endoplasmic reticulum-speciflc apoptosis and triggered the signal-transducing pathways through ER Ca2+ depletion. Two hydrophobic transmembrane domain and ER retention of HAP were required for apoptosis induced by overexpression HAP, which also activate the NFkB. And apoptosis induced by overexpression RTN3/HAP required both ER Ca2+ release and cytosolic Ca2+ elevation, eliciting the activation of caspase-12 and mitochondrial dysfunction through ER Ca2+ depletion. ER calcium release induced by overexpression RTN3/HAP triggered the capacitative (also known as store regulated) calcium entry and the apoptotic signal pathways, such as the activation of procaspase-12, mitochondrial dysfunction.ASY has been identified to can induce apoptosis at a higher effeciency than HAP. The mutants of ASY in which the N-terminal, the C-terminal KK motif or central part was deleted retained full apoptotic ability, indicating that these regions are not required for apoptosis. Surprisingly, a mutant in which the second hydrophobic domain had been deleted, and a mutant with a substitution in the leucine-zipper like motif lost the ability to induce apoptosis, suggesting that the second hydrophobic region, including the leucine-zipper like motif, were important for apoptosis. Except the apoptotic signal pathways of EOR and ER calcium depletion, which triggers the apoptotic signal pathways of overexpression HAP, ASY also mediated apoptosis through another pro-apoptotic pathway CHOP, which may signal death in response to ER stress. This pathway may be a strong correlation with leucine-zipper like motif ofASY.Further we also demonstrated that RTN3/HAP overexpression induced calcium-dependent nitric oxide and inducible nitric oxide synthase. We demonstrated that overexpressed RTN3/HAP and stimuli that activate both EOR and UPR, not UPR only were able to induce up-regulation of nitric oxide synthase. Unlike the apoptosis induced by overexpressed RTN3/HAP required both the ER Ca2+ depletion and the sustained elevation of cytosolic Ca2+, the ER calcium release and ROIs under the early stage of EOR were sufficient to activate the iNOS expression prior to apoptosis. And the NO production and iNOS expression in EOR was demonstrated to be not pro-apoptotic but anti-apoptotic response, via blocking ER Ca2+ release through RyR calcium channel. Our studies suggested that the nitric oxide and inducible nitric oxide synthase represented a likely protective response to endoplasmic reticulum overload response (EOR), not the unfolded protein response (UPR).Though transient high expressed HAP induce apoptosis in several cell lines, such as HeLa, Saos2 and Vero, however, hap and asy was transcribed ubiquitously and RTN3/HAP do not induce apoptosis when transient expression in CHP-100 human neuroepithelioma and stable expression in CHO, Saos-2 and HeLa cells, the intracellular function of ASY and HAP in normal cells and tissues remains to be elucidated. It promotes us to investigate the other function of HAP/ASY rather than apoptosis-inducing.We demonstrated that the DNA binding sites of CHOP/C-EBP, NF-Y and CCAAT box present in the promoter of human, rat and mouse RTN3/HAP, and CHOP and ATF6 were sufficient to up-regulate the expression of RTN3/HAP, suggesting that RTN3/HAP was a target gene of ER stress. Up-regulation of most genes containing potential ER stress element (ERSE) in the promoter regions in response to the UPR were involved in ER protein translocation and transport, it is possible that RTN3/HAP was also involved in ER protein transport.Up-regulation of RTN3/HAP response to the unfolded protein response (UPR) could mediate the increasing Bcl-2 transport to mitochondria. Lowering the expression of RTN3/HAP by RNA interference caused to apoptosis. RTN3/HAP,RTN4B and Bcl-2 could interact with each other, and RTN3/HAP or ASY could block the access of Bcl-2 to the other, the final distribution of Bcl-2 depended on a strong kinetics between RTN3/HAP and ASY. Taken together, our finding provided the highlight that RTN3/HAP played an important role in intracellular trafficking of Bcl-2 and cell survival.We also identified and characterized one sequence encoding the HAP interaction protein from D. melanogaster cDNA library, by using the yeast two-hybrid screen. It encoded a calcium-binding EGF-like domain and was homologous with Fibulin-5. Further we demonstrated that ASY interacted with Fibulin-5 and negatively regulated the secretion of Fibulin-5. ASY was also localized in the surface of HUVEC cells, implying that ASY might be the receptor of Fibulin-5 and mediate the adhesion, migration and expansion of HUVEC cells. Taken together, our finding of the relation between ASY and Fibulin-5 enrich our understanding of cancer therapy and cutis laxa, and diseased adult vasculature.