Death-associated Protein 5 Research Articles

Modeling the structure and DAP5-binding site of the FGF-9 5'-UTR RNA utilized in cap-independent translation.

Cap-independent or eukaryotic initiation factor (eIF) 4E-independent, translation initiation in eukaryotes requires scaffolding protein eIF4G or its homolog, death-associated protein 5 (DAP5). eIF4G associates with the 40S ribosomal subunit, recruiting the ribosome to the RNA transcript. A subset of RNA transcripts, such as fibroblast growth factor 9 (FGF-9), contain 5' untranslated regions (5' UTRs) that directly bind DAP5 or eIF4GI. For viral mRNA, eIF recruitment usually utilizes RNA structure, such as a pseudoknot or stem-loops, and the RNA-helicase eIF4A is required for DAP5- or 4G-mediated translation, suggesting these 5' UTRs are structured. However, for cellular IRES-like translation, no consensus RNA structures or sequences have yet been identified for eIF binding. However, the DAP5-binding site within the FGF-9 5' UTR is unknown. Moreover, DAP5 binds to other, dissimilar 5' UTRs, some of which require an unpaired, accessible 5' end to stimulate cap-independent translation. Using SHAPE-seq, we modeled the 186 nt FGF-9 5'-UTR RNA's complex secondary structure in vitro. Further, DAP5 footprinting, toeprinting, and UV cross-linking experiments identify DAP5-RNA interactions. Modeling of FGF-9 5'-UTR tertiary structure aligns DAP5-interacting nucleotides on one face of the predicted structure. We propose that RNA structure involving tertiary folding, rather than a conserved sequence or secondary structure, acts as a DAP5-binding site. DAP5 appears to contact nucleotides near the start codon. Our findings offer a new perspective in the hunt for cap-independent translational enhancers. Structural, rather than sequence-specific, eIF-binding sites may act as attractive chemotherapeutic targets or as dosage tools for mRNA-based therapies.

Abstract 435: A key regulatory role of the translation initiation factor DAP5 in the metabolic reprogramming of metastatic breast cancer

Abstract Triple negative breast cancer (TNBC) is one of the most aggressive subtypes of breast cancer, accounting for 10-15% of all breast cancers in women. The frequency of metastasis in TNBC is disproportionally high compared to other subtypes of breast cancer and is the primary cause of patient mortality. At the cellular level, TNBC's aggressive nature demands rapid adaptation of the tumor cells to navigate cellular stressors for survival and metastatic transformation. These survival adaptations include a cancer cell’s ability to reprogram its metabolism and regulate mRNA translation control, coordinating alternative pathways to retain a selective proteome and metabolites that will aid in survival, proliferation, and migration. Advanced cancers, including TNBC, are generally highly glycolytic, and have lower rates of mitochondrial respiration (OXPHOS), a phenomenon known as the Warburg effect. This metabolic shift prioritizes the generation of essential metabolic intermediates, to facilitate the production of cellular biomass and fuel proliferation. Another notable alternative pathway is mediated by the ubiquitously expressed translation initiation factor, Death Associated Protein 5 (DAP5). DAP5 is a homolog of eIF4G and mediates translation independently of the canonical mTORC1/eIF4E pathway. Our findings suggest that this alternative translation initiation plays a crucial role in regulating the metabolic reprogramming of metastatic breast cancer. This non-canonical pathway via DAP5 facilitates the selective translation of 20-30% cap-dependent mRNAs, many of which are essential in cancer cell survival, migration, and metabolism. Importantly, it has recently been shown that DAP5 is critical for breast cancer metastasis but not in primary tumor development. Furthermore, TNBC patient data shows that elevated levels of DAP5 mRNA are correlated with poor survival and metastasis. Our data, using the highly metastatic TNBC cell line, MDA-MB-231, demonstrated that silencing DAP5 significantly reduced glycolytic rate and glycolytic capacity. Consistent with these findings, translatome profiling, showed that DAP5 silencing reduced numerous metabolic targets including glycolytic enzymes and metabolite transporters. Calcium (Ca2+) signaling, which has an important role in cancer progression, migration, and cellular bioenergetics is also affected by DAP5 in TNBC cells. Collectively, our findings indicate that breast cancer cells utilize the translation initiation factor DAP5 to selectively translate mRNAs that support metabolic reprogramming and cell migration, suggesting that DAP5 has a key regulatory role in the cancer’s transformation. Citation Format: Erna Mitaishvili, Guilherme Henrique Souza Bomfim, Anna Zou, Moira Sauane, Rodrigo Lacruz, Columba de la Parra. A key regulatory role of the translation initiation factor DAP5 in the metabolic reprogramming of metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 435.

High expression of death-associated protein 5 promotes glucose metabolism in gastric cancer cells and correlates with poor survival outcomes

To investigate the prognostic value of death-associated protein 5 (DAP5) in gastric cancer (GC) and its regulatory effect on aerobic glycolysis in GC cells. We analyzed DAP5 expression levels in GC and adjacent tissues and its association with survival outcomes of GC patients using public databases. We collected paired samples of GC and adjacent tissues from 102 patients undergoing radical resection of GC in our hospital from June, 2012 to July, 2017, and analyzed the correlation of DAP5 expression level detected immunohistochemically with the clinicopathological parameters of the patients. Cox regression analysis, Kaplan-Meier analysis, and ROC curves were used to explore the independent risk factors and the predictive value of DAP5 expression for 5-year survival of the patients. In the cell experiments, we observed the changes in aerobic glycolysis in MGC-803 cells following lentivirus-mediated DAP5 knockdown or overexpression by measuring glucose uptake and cellular lactate level and using qRT-PCR and Western blotting. Analysis using the public databases showed that DAP5 was highly expressed in GC and correlated with tumor progression and poor survival outcomes of the patients (P < 0.05). In the clinical samples, DAP5 expression was significantly higher in GC than in the adjacent tissues (3.19±0.60 vs 1.00±0.12; t=36.863, P < 0.01), and a high expression of DAP5 was associated with a reduced 5-year survival rate of the patients (17.6% vs 72.5%; χ2=29.921, P < 0.05). A high DAP5 expression, T3-4, N2-3, and CEA≥5 ng/mL were identified as independent risk factors affecting 5-year survival outcomes of GC (P < 0.05), for which DAP5 expression showed a prediction sensitivity, specificity and accuracy of 73.2%, 80.4% and 79.0%, respectively. In MGC-803 cells, DAP5 knockdown significantly reduced glucose uptake, lactate level and the expressions of GLUT1, HK2 and LDHA, and DAP5 overexpression produced the opposite effects (P < 0.05). A high expression of DAP5 in GC, which enhances cellular aerobic glycolysis to promote cancer progression, is correlated with a poor survival outcome and may serve as a biomarker for evaluating long-term prognosis of GC patients.

5′-UTR recruitment of the translation initiation factor eIF4GI or DAP5 drives cap-independent translation of a subset of human mRNAs

During unfavorable conditions (e.g. tumor hypoxia or viral infection), canonical, cap-dependent mRNA translation is suppressed in human cells. Nonetheless, a subset of physiologically important mRNAs (e.g. hypoxia-inducible factor 1α [HIF-1α], fibroblast growth factor 9 [FGF-9], and p53) is still translated by an unknown, cap-independent mechanism. Additionally, expression levels of eukaryotic translation initiation factor 4GI (eIF4GI) and of its homolog, death-associated protein 5 (DAP5), are elevated. By examining the 5' UTRs of HIF-1α, FGF-9, and p53 mRNAs and using fluorescence anisotropy binding studies, luciferase reporter-based in vitro translation assays, and mutational analyses, we demonstrate here that eIF4GI and DAP5 specifically bind to the 5' UTRs of these cap-independently translated mRNAs. Surprisingly, we found that the eIF4E-binding domain of eIF4GI increases not only the binding affinity but also the selectivity among these mRNAs. We further demonstrate that the affinities of eIF4GI and DAP5 binding to these 5' UTRs correlate with the efficiency with which these factors drive cap-independent translation of these mRNAs. Integrating the results of our binding and translation assays, we conclude that eIF4GI or DAP5 is critical for recruitment of a specific subset of mRNAs to the ribosome, providing mechanistic insight into their cap-independent translation.

DAP5 increases axonal outgrowth of hippocampal neurons by enhancing the cap-independent translation of DSCR1.4 mRNA

Proper wiring between neurons is indispensable for proper brain function. From the early developmental stage, axons grow and navigate to connect to targets according to specific guidance cues. The accuracy of axonal outgrowth and navigation are controlled by a variety of genes, and mutations and/or deficiencies in these genes are closely related to several brain disorders, such as autism. DSCR1 is one of these genes and regulates actin filament formation in axons. Thus, identifying the detailed regulatory mechanisms of DSCR1 expression is crucial for the understanding of the axon development of neurons; however, these regulatory mechanisms of DSCR1 remain unknown. Here, we discovered that mRNA encoding the DSCR1 isoform DSCR1.4 is present and mainly translated by the cap-independent initiation mechanisms in both the soma and axons of hippocampal neurons. We found that translation of DSCR1.4 mRNA is enhanced by death-associated protein 5 (DAP5), which can bind to DSCR1.4 5′UTR. BDNF-stimulus induced an increase in DAP5 expression and the cap-independent translation efficiency of DSCR1.4 mRNA in axon as well as soma. Furthermore, we showed the importance of the cap-independent translation of DSCR1.4 on enhancement of DSCR1.4 expression by BDNF-stimulus and axonal outgrowth of hippocampal neurons. Our findings suggest a new translational regulatory mechanism for DSCR1.4 expressions and a novel function of DAP5 as a positive regulator of DSCR1.4 mRNA translation induced in soma and axon of hippocampal neurons.

YelA, a putative Dictyostelium translational regulator, acts as antagonist of DIF-1 signaling to control cell-type proportioning.

DIF-1 (differentiation-inducing factor1) is a polyketide produced by Dictyostelium prespore cells which induces initially uncommitted cells to differentiate as prestalk cells. Exposure of cells to DIF-1 causes transitory hypo-phosphorylation of seven serine residues in YelA, a protein with a region of strong homology to the MIF4G domain of the eukaryotic initiation factor eIF4G. Based upon its domain architecture, which in one important aspect closely resembles that of Death-Associated Protein 5 (DAP5), we predict a role in stimulating internal ribosome entry-driven mRNA translation. The two paradigmatic DIF-1 inducible genes are ecmA (extracellular matrixA) and ecmB. In support of a YelA function in DIF-1 signaling, a YelA null strain showed greatly increased expression of ecmA and ecmB in response to DIF-1. Also, during normal development in the null strain, expression of the two genes is accelerated. This is particularly evident for ecmB, a marker of stalk tube and supporting structure differentiation. Mutants in DIF-1 bio-synthesis or signaling display a rudimentary or no basal disc and, conversely, YelA null mutants produce fruiting bodies with a highly enlarged basal disc that ectopically expresses a stalk tube-specific marker. Thus YelA acts as an antagonist of DIF-1 signaling, with a consequent effect on cell type proportioning and it is predicted to act as a translational regulator.

Cleavage of DAP5 by coxsackievirus B3 2A protease facilitates viral replication and enhances apoptosis by altering translation of IRES-containing genes.

Cleavage of eukaryotic translation initiation factor 4G (eIF4G) by enterovirus proteases during infection leads to the shutoff of cellular cap-dependent translation, but does not affect the initiation of cap-independent translation of mRNAs containing an internal ribosome entry site (IRES). Death-associated protein 5 (DAP5), a structural homolog of eIF4G, is a translation initiation factor specific for IRES-containing mRNAs. Coxsackievirus B3 (CVB3) is a positive single-stranded RNA virus and a primary causal agent of human myocarditis. Its RNA genome harbors an IRES within the 5'-untranslated region and is translated by a cap-independent, IRES-driven mechanism. Previously, we have shown that DAP5 is cleaved during CVB3 infection. However, the protease responsible for cleavage, cleavage site and effects on the translation of target genes during CVB3 infection have not been investigated. In the present study, we demonstrated that viral protease 2A but not 3C is responsible for DAP5 cleavage, generating 45- and 52-kDa N- (DAP5-N) and C-terminal (DAP5-C) fragments, respectively. By site-directed mutagenesis, we found that DAP5 is cleaved at amino acid G434. Upon cleavage, DAP5-N largely translocated to the nucleus at the later time points of infection, whereas the DAP5-C largely remained in the cytoplasm. Overexpression of these DAP5 truncates demonstrated that DAP5-N retained the capability of initiating IRES-driven translation of apoptosis-associated p53, but not the prosurvival Bcl-2 (B-cell lymphoma 2) when compared with the full-length DAP5. Similarly, DAP5-N expression promoted CVB3 replication and progeny release; on the other hand, DAP5-C exerted a dominant-negative effect on cap-dependent translation. Taken together, viral protease 2A-mediated cleavage of DAP5 results in the production of two truncates that exert differential effects on protein translation of the IRES-containing genes, leading to enhanced host cell death.

Cleavage of DAP5 by coxsackievirus B3 protease 2A causes its nuclear translocation and inhibition of IRES‐containing gene transcription (836.5)

Coxsackievirus B3 (CVB3) is a primary causative agent of viral myocarditis which may progress to dilated cardiomyopathy (DCM), a leading cause of unexpected death in children and young adults. Upon infection, CVB3 hijacks host cellular translation machinery, generating an environment that favors viral replication. Death associated protein 5 (DAP5) is an internal ribosomal entry site (IRES) specific translation initiation factor, utilized by the cell in conditions of stress, differentiation, cell‐cycle and apoptosis. We previously showed that DAP5 is cleaved during CVB3 infection. In this study we aimed to identify the responsible protease and site of cleavage, as well as the functional impact. Our results found that DAP5 was cleaved by viral protease 2A but not by cellular proteases. We further identified the cleavage site at amino acid G434. Upon cleavage, the N‐terminal product could translocate to the nucleus and inhibited transcription of IRES‐containing pro‐survival genes such as Bcl‐2, GRP‐78 and VEGF. This is the first report to reveal the function and mechanism of DAP5 cleavage in regulating viral and host gene expression during CVB3 infection. Moreover, this study may identify key components necessary for facilitating viral pathogenesis and thus provide novel pharmaceutical targets for viral myocarditis.Grant Funding Source: Canadian Institutes of Health Research

DAP5 Ameliorates Cisplatin-Induced Apoptosis of Renal Tubular Cells

Background: Nephrotoxicity of cisplatin limits its clinical application. Cisplatin-induced acute renal tubular epithelial cell apoptosis is one of the major mechanisms of cisplatin nephrotoxicity. Here, the role and regulation of death-associated protein 5 (DAP5) in cisplatin-induced tubular cell apoptosis were investigated. Methods: After upregulation of DAP5 expression by plasmid transfection and downregulation of DAP5 expression by small interfering RNA in human kidney tubular epithelial cell line (HKC) cells, the degree of cell apoptosis was assessed by flow cytometric analysis. The expression of Bax and Bcl-2 proteins was detected by Western blot analysis. The relationship between the PI3K/Akt/mTOR signaling pathway and DAP5 was also evaluated. Results: During cisplatin-induced apoptosis in HKC cells, DAP5 underwent proteolytic fragmentation, yielding an 86-kDa species, DAP5/p86. Overexpression of DAP5/p97 and DAP5/p86 increased the translation of Bcl-2 and reduced the extent of cisplatin-induced apoptosis. Knockdown of DAP5 expression using small interfering RNA decreased the translation of Bcl-2 and increased the degree of apoptosis. Neither manipulation affected the expression of Bax. DAP5 expression was positively regulated by the PI3K/Akt/mTOR signaling pathway. Conclusion: Collectively, the results from the present study revealed a new role for DAP5 in cisplatin-induced apoptosis: DAP5/p97 and DAP5/p86 enhanced the translation of the anti-apoptotic protein Bcl-2 and inhibited cisplatin-induced apoptosis. The PI3K/Akt/mTOR signaling pathway may positively regulate the expression of DAP5.

Crystallization and preliminary X-ray diffraction analysis of the MIF4G domain of DAP5

Death-associated protein 5 (DAP5) is a member of the eIF4G family of scaffolding proteins that mediate cap-independent translation initiation by recruiting the translational machinery to internal ribosomal entry sites (IRESs) on mRNA. The MIF4G domain of DAP5 directly interacts with the eukaryotic initiation factors eIF4A and eIF3 and enhances the translation of several viral and cellular IRESs. Here, the crystallization and preliminary X-ray diffraction analysis of the MIF4G domain of DAP5 is presented.

Death-associated protein 5 (DAP5/p97/NAT1) contributes to retinoic acid-induced granulocytic differentiation and arsenic trioxide-induced apoptosis in acute promyelocytic leukemia.

All-trans-retinoic acid (ATRA) and arsenic trioxide (ATO) induce differentiation and apoptosis in acute promyelocytic leukemia (APL) cells. Here we investigated the role and regulation of death-associated protein-5 (DAP5/p97/NAT1), a novel inhibitor of translational initiation, in APL cell differentiation and apoptosis. We found that ATRA markedly induced DAP5/p97 protein and gene expression and nuclear translocation during terminal differentiation of APL (NB4) and HL60 cells but not differentiation-resistant cells (NB4.R1 and HL60R), which express very low levels of DAP5/p97. At the differentiation inducing concentrations, ATO (<0.5 microM), dimethyl sulfoxide, 1,25-dihydroxy-vitamin-D3, and phorbol-12-myristate 13-acetate also significantly induced DAP5/p97 expression in NB4 cells. However, ATO administered at apoptotic doses (1-2 microM) induced expression of DAP5/p86, a proapoptotic derivative of DAP5/p97. ATRA and ATO-induced expression of DAP5/p97 was associated with inhibition of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Furthermore, DAP5/p97 expression was upregulated by inhibition of the PI3K/Akt/mammalian target of rapamycin (mTOR) pathway via LY294002 and via rapamycin. Finally, knockdown of DAP5/p97 expression by small interfering RNA inhibited ATRA-induced granulocytic differentiation and ATO-induced apoptosis. Together, our data reveal new roles for DAP5/p97 in ATRA-induced differentiation and ATO-induced apoptosis in APL and suggest a novel regulatory mechanism by which PI3K/Akt/mTOR pathway inhibition mediates ATRA- and ATO-induced expression of DAP5/p97.

Novel Tumor Suppressor Protein Programmed Cell Death 4 (PDCD4) Suppresses Activity of PI3K/Akt Pathway and Regulates Expression of p27 (Kip1) and c-myc, DAP5 and Willm's Tumor (WT1) in Acute Myeloid Leukemia.

Programmed cell death 4 (PDCD4) is a recently identied novel tumor suppressor protein that inhibits cap-dependent mRNA translation. PDCD4 inhibits tumor promoter incuced carcinogenesis and transformation by suppressing the helicase activity of eIF4A, leading to translational inhibition. Recently we found that PDCD4 is required for all-trans-retinoic acid (ATRA)-induced granulocytic differentiation of acute promyelocytic leukemia (APL) cells (Ozpolat&Akar et al, Mol Cancer Res, in press), type of acute myeloid leukemia characterized by a t(15;17) and a differentiation block. Here we investigated the downstream mediators or targets of PDCD4 in leukemia cell differentiation. ATRA is currently used as a first line standard treatment in APL. Recently, we reported that ATRA induces translational suppression through multiple posttranscriptional mechanisms that involve suppression of translation initiation, a rate limiting step of protein synthesis (Harris&Ozpolat et al, Blood , 104 (5) 2004). We found that ATRA treatment induced PDCD4 expression in NB4 APL, HL60 AML, primary APL patient leukemia cells and normal human CD34+ bone marrow progenitors cells during granulocytic differentiation. However, ATRA/maturation resistant NB4.R1 and HL60R cells failed to express and translocate PDCD4 into nucleus after ATRA treatment. To identify downstream targets of PDCD4 we knock downed PDCD4 expression by siRNA and examined changes in the expression of target proteins that are known to be regulated by ATRA by Western blot analysis in NB4 cells. We found that PDCD4 represses c-myc, and WT1 expression however, it is required for the expression of DAP5 (death associated protein 5), and cyclin dependent kinase inhibitor p27KIP1, and but not c-jun, p21Cip1, and tissue transglutaminase (TG2). Inhibition of PDCD4 by siRNA resulted in upregulation of phospho-P70S6K, suggesting that PDCD4 inhibits activity of PI3K/Akt pathway. RT-PCR analysis revealed that mRNA of these proteins did not change suggesting that PDCD4 tumor suppressor protein regulates expression of these important cellular proteins at translational level and suppresses PI3K/Akt pathway. Furthermore we rapamycin, a specific mTOR inhibitor currently in clinical trials in AML, induced a marked expression of PDCD4, which regulates c-myc and p27 Kip1, revealing a novel mechanism of action of rapamycin, providing new rationale for targeting translational pathways as a therapeutic intervention in the treatment of AML. Overall, data suggest that PDCD4 regulates expression of critical cellular proteins involved in differentiation of leukemia cells and PDCD4 mediated translational control may be an important regulatory mechanism for regulation of gene expression.

DAP5 and IRES-mediated translation during programmed cell death

DAP5 (Death Associated Protein 5), also named p97 and NAT1, is a member of the eIF4G family that lacks the eIF4E binding site. Its function was linked to programmed cell death (PCD) based on the seminal finding that a fragment of DAP5/p97 protein, which acted in a dominant-negative manner, protected against IFN-gamma (IFN-)-induced cell death. Subsequently, it was found that DAP5 protein is activated during cell death by caspase cleavage, yielding a C-terminal-truncated protein of 86 kDa. The p86 form promotes internal ribosome entry site (IRES)-dependent translation of several mRNAs including Apaf-1, c-myc, XIAP, HIAP2 and DAP5 itself, which carries an IRES element in its 5'UTR. The activation of DAP5 IRES creates a positive feedback loop, which results in sustained translation of DAP5 protein during PCD under conditions of reduced cap-dependent translation. DAP5 deficiency prevents embryonic stem (ES) cell differentiation, suggesting a wider range of DAP5 mRNA targets and additional mechanisms that might activate the protein.

The caspase-cleaved DAP5 protein supports internal ribosome entry site-mediated translation of death proteins.

Apoptosis is characterized by a translation switch from cap-dependent to internal ribosome entry site (IRES)-mediated protein translation. During apoptosis, several members of the eukaryotic initiation factor (eIF)4G family are cleaved specifically by caspases. Here we investigated which of the caspase-cleaved eIF4G family members could support cap-independent translation through IRES elements that retain activity in the dying cell. We focused on two major fragments arising from the cleavage of eIF4GI and death-associated protein 5 (DAP5) proteins (eIF4GI M-FAG/p76 and DAP5/p86, respectively), because they are the only potential candidates to preserve the minimal scaffold function needed to mediate translation. Transfection-based experiments in cell cultures indicated that expression of DAP5/p86 in cells stimulated protein translation from the IRESs of c-Myc, Apaf-1, DAP5, and XIAP. In contrast, these IRESs were refractory to the ectopically expressed eIF4GI M-FAG/p76. Furthermore, our study provides in vivo evidence that the caspase-mediated removal of the C-terminal tail of DAP5/p97 relieves an inhibitory effect on the protein's ability to support cap-independent translation through the DAP5 IRES. Altogether, the data suggest that DAP5 is a caspase-activated translation factor that mediates translation through a repertoire of IRES elements, supporting the translation of apoptosis-related proteins.

Up-regulation of PDCD4 in senescent human diploid fibroblasts

Programmed cell death 4 (PDCD4) has a common MI domain sharing with death associated protein 5 (DAP5) and a component of eukaryotic translation initiation factor (eIF4G) complex and it might also work as a tumor suppressor. We could find that the message and product of Pdcd4 gene were up-regulated in senescent human diploid fibroblasts. In yeast two hybrid analysis, the C-terminal region of PDCD4 interacted with ribosomal protein S13 (RPS13), ribosomal protein L5 (RPL5), and TI-227H. In in vitro binding assay, RPS13, a component of 40S ribosome was stably bound to PDCD4. We also found that PDCD4 was localized to polysome fractions. We could pull out eIF4G with GST-PDCD4, but eIF4E did not interact with PDCD4. From these results, we could assume that PDCD4 might regulate the eIF4G-dependent translation through direct interactions with eIF4G and RPS13 in senescent fibroblasts.

Molecular pathways of protein synthesis inhibition during brain reperfusion: implications for neuronal survival or death.

Protein synthesis inhibition occurs in neurons immediately on reperfusion after ischemia and involves at least alterations in eukaryotic initiation factors 2 (eIF2) and 4 (eIF4). Phosphorylation of the alpha subunit of eIF2 [eIF2(alphaP)] by the endoplasmic reticulum transmembrane eIF2alpha kinase PERK occurs immediately on reperfusion and inhibits translation initiation. PERK activation, along with depletion of endoplasmic reticulum Ca2+ and inhibition of the endoplasmic reticulum Ca2+ -ATPase, SERCA2b, indicate that an endoplasmic reticulum unfolded protein response occurs as a consequence of brain ischemia and reperfusion. In mammals, the upstream unfolded protein response components PERK, IRE1, and ATF6 activate prosurvivial mechanisms (e.g., transcription of GRP78, PDI, SERCA2b ) and proapoptotic mechanisms (i.e., activation of Jun N-terminal kinases, caspase-12, and CHOP transcription). Sustained eIF2(alphaP) is proapoptotic by inducing the synthesis of ATF4, the CHOP transcription factor, through "bypass scanning" of 5' upstream open-reading frames in ATF4 messenger RNA; these upstream open-reading frames normally inhibit access to the ATF4 coding sequence. Brain ischemia and reperfusion also induce mu-calpain-mediated or caspase-3-mediated proteolysis of eIF4G, which shifts message selection to m 7 G-cap-independent translation initiation of messenger RNAs containing internal ribosome entry sites. This internal ribosome entry site-mediated translation initiation (i.e., for apoptosis-activating factor-1 and death-associated protein-5) can also promote apoptosis. Thus, alterations in eIF2 and eIF4 have major implications for which messenger RNAs are translated by residual protein synthesis in neurons during brain reperfusion, in turn constraining protein expression of changes in gene transcription induced by ischemia and reperfusion. Therefore, our current understanding shifts the focus from protein synthesis inhibition to the molecular pathways that underlie this inhibition, and the role that these pathways play in prosurvival and proapoptotic processes that may be differentially expressed in vulnerable and resistant regions of the reperfused brain.

DAP-5 is involved in MycN/IFNγ-induced apoptosis in human neuroblastoma cells

Death associated protein-5 (DAP-5) is a ubiquitously expressed member of the translation initiation factor eIF4G family that lacks the eIF4E binding site. A dominant negative fragment of DAP-5 protects HeLa cells from IFNγ-induced cell death. By employing a functional approach we examined the role of DAP-5 in human neuroblastoma cells that are sensitized for IFNγ-induced apoptosis by tetracycline controlled MYCN expression. DAP-5 fragment transcribed at high levels and translated into a functional miniprotein of 28 kDa protected neuroblastoma cells from IFNγ-induced apoptosis. Reduced serum levels were toxic to cells constitutively expressing DAP-5 fragment suggesting that DAP-5 protein is essential for both viability and death of human neuroblastoma cells.