scholarly journals Complex formation between deoxyhypusine synthase and its protein substrate, the eukaryotic translation initiation factor 5A (eIF5A) precursor

1999 ◽  
Vol 340 (1) ◽  
pp. 273-281 ◽  
Author(s):  
Young Bok LEE ◽  
Young Ae JOE ◽  
Edith C. WOLFF ◽  
Emilios K. DIMITRIADIS ◽  
Myung Hee PARK
Keyword(s):  
Active Sites ◽  
Analytical Ultracentrifugation ◽  
Cellular Protein ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Stable Complex ◽  
Deoxyhypusine Synthase ◽  
Mobility Shift ◽  
Protein Substrate ◽  
Equilibrium Ultracentrifugation

Deoxyhypusine synthase catalyses the first step in the post-translational synthesis of hypusine [Nϵ-(4-amino-2-hydroxybutyl) lysine] in a single cellular protein, the precursor of eukaryotic initiation factor 5A (eIF5A). Deoxyhypusine synthase exists as a tetramer with four potential active sites. The formation of a stable complex between human deoxyhypusine synthase and its protein substrate, human recombinant eIF5A precursor (ec-eIF5A), was examined by affinity chromatography using polyhistidine-tagged (His·Tag) ec-eIF5A, by a gel mobility-shift method, and by analytical ultracentrifugation. Deoxyhypusine synthase was selectively retained by His·Tag-ec-eIF5A immobilized on a resin. The complex of deoxyhypusine synthase and ec-eIF5A was separated from the free enzyme and protein substrate by electrophoresis under non-denaturing conditions. The stoichiometry of the two components in the complex was estimated to be 1 deoxyhypusine synthase tetramer to 1 ec-eIF5A monomer by N-terminal amino acid sequencing of the complex. Equilibrium ultracentrifugation data further supported this 1:1 ratio and indicated a very strong interaction of the enzyme with ec-eIF5A (Kd≤ 0.5 nM). Formation of the complex was not dependent on NAD+ or spermidine and occurred at pH 7.0-9.2. An enzyme-product complex, as well as the deoxyhypusine-containing product (modified ec-eIF5A), was also detected at pH 7.0-9.2 in a complete reaction mixture containing 1 mM spermidine.

scholarly journals Human deoxyhypusine synthase: interrelationship between binding of NAD and substrates

2000 ◽  
Vol 352 (3) ◽  
pp. 851-857 ◽  
Author(s):  
Chang Hoon LEE ◽  
Myung Hee PARK
Keyword(s):  
Reaction Mechanism ◽  
Conformational Change ◽  
Translation Initiation ◽  
Binding Sites ◽  
Cellular Protein ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Deoxyhypusine Synthase ◽  
Eukaryotic Translation ◽  
Eukaryotic Translation Initiation

Deoxyhypusine synthase catalyses the NAD-dependent transfer of the butylamine moiety from the polyamine, spermidine, to a specific lysine residue of a single cellular protein, eukaryotic translation-initiation factor 5A (eIF5A) precursor. The native enzyme exists as a tetramer of four identical subunits and contains four binding sites for NAD. The binding of spermidine and NAD was studied by a filtration assay. [3H]Spermidine binding to the enzyme was not detectable alone or in the presence of the eIF5A precursor, but was detected only in the presence of NAD or NADH, suggesting that a NAD/NADH-induced conformational change is required for the binding of spermidine. A strong NAD-dependent binding was also observed with a spermidine analogue, N1-guanyl-1,7-diamino[3H]heptane (GC7), but not with [14C]putrescine or [14C]spermine. Although [3H]NAD binding to the enzyme occurred in the absence of spermidine, its affinity for the enzyme was markedly enhanced by spermidine, GC7 and also by the eIF5A precursor. The maximum binding for NAD and spermidine was estimated to be ≈ 4 molecules each/enzyme tetramer. The dependence of spermidine binding on NAD and the modulation of binding of NAD by spermidine and the eIF5A precursor suggest intricate relationships between the binding of cofactor and the substrates, and provide new insights into the reaction mechanism.

scholarly journals Nuclear Localization of Cytoplasmic Poly(A)-Binding Protein upon Rotavirus Infection Involves the Interaction of NSP3 with eIF4G and RoXaN

2008 ◽  
Vol 82 (22) ◽  
pp. 11283-11293 ◽  
Author(s):  
Maya Harb ◽  
Michelle M. Becker ◽  
Damien Vitour ◽  
Carolina H. Baron ◽  
Patrice Vende ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Translation Initiation ◽  
Nuclear Export ◽  
Nonstructural Protein ◽  
Binding Protein ◽  
Nuclear Export Signal ◽  
Cellular Protein ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Viral Mrnas

ABSTRACT Rotavirus nonstructural protein NSP3 interacts specifically with the 3′ end of viral mRNAs, with the eukaryotic translation initiation factor eIF4G, and with RoXaN, a cellular protein of yet-unknown function. By evicting cytoplasmic poly(A) binding protein (PABP-C1) from translation initiation complexes, NSP3 shuts off the translation of cellular polyadenylated mRNAs. We show here that PABP-C1 evicted from eIF4G by NSP3 accumulates in the nucleus of rotavirus-infected cells. Through modeling of the NSP3-RoXaN complex, we have identified mutations in NSP3 predicted to interrupt its interaction with RoXaN without disturbing the NSP3 interaction with eIF4G. Using these NSP3 mutants and a deletion mutant unable to associate with eIF4G, we show that the nuclear localization of PABP-C1 not only is dependent on the capacity of NSP3 to interact with eIF4G but also requires the interaction of NSP3 with a specific region in RoXaN, the leucine- and aspartic acid-rich (LD) domain. Furthermore, we show that the RoXaN LD domain functions as a nuclear export signal and that RoXaN tethers PABP-C1 with RNA. This work identifies RoXaN as a cellular partner of NSP3 involved in the nucleocytoplasmic localization of PABP-C1.

scholarly journals The role of hypusine depletion in cytostasis induced by S-adenosyl-l-methionine decarboxylase inhibition: new evidence provided by 1-methylspermidine and 1,12-dimethylspermine

1994 ◽  
Vol 303 (2) ◽  
pp. 363-368 ◽  
Author(s):  
T L Byers ◽  
J R Lakanen ◽  
J K Coward ◽  
A E Pegg
Keyword(s):  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Decarboxylase Inhibitor ◽  
Deoxyhypusine Synthase ◽  
Eukaryotic Translation ◽  
Polyamine Analogues ◽  
L1210 Cells ◽  
Eukaryotic Translation Initiation ◽  
New Evidence

The abilities of the natural polyamines, spermidine and spermine, and of the synthetic analogues, 1-methylspermidine and 1,12-dimethylspermine, to reverse the effects of the S-adenosyl-L-methionine decarboxylase inhibitor 5′-([(Z)-4-aminobut-2-enyl]methylamino)-5′-deoxyadenosine (AbeAdo) on L1210-cell growth were studied. L1210 cells were exposed to AbeAdo for 12 days to induce cytostasis and then exposed to spermidine, spermine, 1-methylspermidine or 1,12-dimethylspermine in the continued presence of AbeAdo. AbeAdo-induced cytostasis was overcome by the natural polyamines, spermidine and spermine. The cytostasis was also reversed by 1-methylspermidine. 1,12-Dimethylspermine had no effect on the AbeAdo-induced cytostasis of chronically treated cells, although it was active in permitting growth of cells treated with the ornithine decarboxylase inhibitor, alpha-difluoromethylornithine. The initial 12-day exposure to AbeAdo elevated intracellular putrescine levels, depleted intracellular spermidine and spermine, and resulted in the accumulation of unmodified eukaryotic translation initiation factor 5A (eIF-5A). Exposure of these cells to exogenous spermidine, which is the natural substrate for deoxyhypusine synthase, resulted in a decrease in the unmodified eIF-5A content. 1-Methylspermidine, which was found to be a substrate of deoxyhypusine synthase in vitro, also decreased the levels of unmodified eIF-5A in the AbeAdo-treated cells. Although spermine is not a substrate of deoxyhypusine synthase, spermine was converted into spermidine in the L1210 cells, and spermine addition to AbeAdo-treated cells resulted in the appearance of both intracellular spermine and spermidine and in the decrease in unmodified eIF-5A. Exogenous 1,12-dimethylspermine, which was not metabolized to spermine or to 1-methylspermidine and was not a substrate of deoxyhypusine synthase in vitro, did not decrease levels of unmodified eIF-5A. The finding that AbeAdo-induced cytostasis was only reversed by polyamines and polyamine analogues that result in the formation of hypusine or an analogue in eIF-5A is consistent with the hypothesis [Byers, Wiest, Wechter and Pegg (1993) Biochem. J. 290, 115-121] that AbeAdo-induced cytostasis is due to the depletion of the hypusine-containing form of eIF-5A, which is secondary to the depletion of spermidine by inhibition of S-adenosyl-L-methionine decarboxylase.

scholarly journals Eukaryotic translation initiation factor 5A inhibition alters physiopathology and immune responses in a “humanized” transgenic mouse model of type 1 diabetes

2014 ◽  
Vol 306 (7) ◽  
pp. E791-E798 ◽  
Author(s):  
Shahnawaz Imam ◽  
Raghavendra G. Mirmira ◽  
Juan C. Jaume
Keyword(s):  
T Cells ◽  
Type 1 Diabetes ◽  
Translation Initiation ◽  
Immune Modulation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
T Regulatory Cell ◽  
Deoxyhypusine Synthase ◽  
Diabetes Outcome

Therapeutic options for treatment of type 1 diabetes (T1D) are still missing. New avenues for immune modulation need to be developed. Here we attempted at altering the diabetes outcome of our humanized model of T1D by inhibiting translation-initiation factor eIF5A hypusination in vivo. Double-transgenic (DQ8-GAD65) mice were immunized with adenoviral vectors carrying GAD65 for diabetes induction. Animals were subsequently treated with deoxyhypusine synthase (DHS) inhibitor GC7 and monitored for diabetes development over time. On one hand, helper CD4+ T cells were clearly affected by the downregulation of the eIF5A not just at the pancreas level but overall. On the other hand, the T regulatory cell component of CD4 responded with activation and proliferation significantly higher than in the non-GC7-treated controls. Female mice seemed to be more susceptible to these effects. All together, our results show for the first time that downregulation of eIF5A through inhibition of DHS altered the physiopathology and observed immune outcome of diabetes in an animal model that closely resembles human T1D. Although the development of diabetes could not be abrogated by DHS inhibition, the immunomodulatory capacity of this approach may supplement other interventions directed at increasing regulation of autoreactive T cells in T1D.

Caspases are not involved in the cleavage of translation initiation factor eIF4GI during picornavirus infection

Microbiology ◽  
2000 ◽  
Vol 81 (7) ◽  
pp. 1703-1707 ◽  
Author(s):  
Lisa O. Roberts ◽  
Angela J. Boxall ◽  
Louisa J. Lewis ◽  
Graham J. Belsham ◽  
George E. N. Kass
Keyword(s):  
Translation Initiation ◽  
Caspase 3 ◽  
Foot And Mouth Disease ◽  
Cellular Protein ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Fmdv Infection ◽  
Expression Studies ◽  
Mouth Disease Virus ◽  
Picornavirus Infection

Infection of cells by many picornaviruses results in the rapid inhibition of cellular protein synthesis due to cleavage of the translation initiation factor eIF4G. The poliovirus (PV) 2A and foot-and-mouth disease virus (FMDV) L proteases are each sufficient to mediate this cleavage, but the cleavage mechanism may be indirect, involving an unidentified cellular protease(s). eIF4G is also targetted for cleavage by caspase-3 during apoptosis. Here, it is shown that caspase inhibitors do not inhibit the cleavage of eIF4GI during PV or FMDV infection. Similarly, in transient-expression studies, the cleavage of eIF4GI induced by PV 2A or FMDV L was unaffected by these inhibitors. Furthermore, the cleavage of eIF4GI was observed in PV-infected MCF-7 cells lacking caspase-3. These data, and the fact that induction of apoptosis yields different eIF4GI cleavage fragments, indicate that caspases do not have a major role in the cleavage of eIF4GI during PV or FMDV infection.

scholarly journals Independent roles of eIF5A and polyamines in cell proliferation

2005 ◽  
Vol 385 (3) ◽  
pp. 779-785 ◽  
Author(s):  
Kazuhiro NISHIMURA ◽  
Kaori MUROZUMI ◽  
Akira SHIRAHATA ◽  
Myung Hee PARK ◽  
Keiko KASHIWAGI ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Growth ◽  
Translation Initiation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Deoxyhypusine Synthase ◽  
Eukaryotic Translation ◽  
Spermine Synthase ◽  
Polyamine Content ◽  
Eukaryotic Translation Initiation

To examine the roles of active hypusinated eIF5A (eukaryotic translation initiation factor 5A) and polyamines in cell proliferation, mouse mammary carcinoma FM3A cells were treated with an inhibitor of deoxyhypusine synthase, GC7 (N1-guanyl-1, 7-diaminoheptane), or with an inhibitor of ornithine decarboxylase, DFMO (α-difluoromethylornithine), or with DFMO plus an inhibitor of spermine synthase, APCHA [N1-(3-aminopropyl)-cyclohexylamine]. Treatment with GC7 decreased the level of active eIF5A on day 1 without affecting cellular polyamine content, and inhibition of cell growth occurred from day 2. This delay reflects the fact that eIF5A was present in excess and was very stable in these cells. Treatment with DFMO or with DFMO plus APCHA inhibited cell growth on day 1. DFMO considerably decreased the levels of putrescine and spermidine, and the formation of active eIF5A began to decrease when the level of spermidine fell below 8 nmol/mg of protein after 12 h of incubation with DFMO. The combination of DFMO and APCHA markedly decreased the levels of putrescine and spermine and significantly decreased the level of spermidine, but did not affect the level of active eIF5A until day 3 when spermidine level decreased to 7 nmol/mg of protein. The results show that a decrease in either active eIF5A or polyamines inhibits cell growth, indicating that eIF5A and polyamines are independently involved in cell growth.

scholarly journals Identification of mRNA that binds to eukaryotic initiation factor 5A by affinity co-purification and differential display

2004 ◽  
Vol 384 (3) ◽  
pp. 585-590 ◽  
Author(s):  
Aiguo XU ◽  
David Li-En JAO ◽  
Kuang Yu CHEN
Keyword(s):  
Differential Display ◽  
Initiation Factor ◽  
Eukaryotic Initiation Factor ◽  
Deoxyhypusine Synthase ◽  
Mobility Shift ◽  
Arbitrary Primers ◽  
Stem Loop ◽  
Deoxyhypusine Hydroxylase ◽  
Rev Response Element

Eukaryotic initiation factor 5A (eIF-5A) is the only protein in nature that contains hypusine, an unusual amino acid formed post-translationally by deoxyhypusine synthase and deoxyhypusine hydroxylase. Genetic and pharmacological evidence suggests that eIF-5A is essential for cell survival and proliferation. However, the precise function and interacting partners of eIF-5A remain unclear. We have shown previously that eIF-5A can bind to RRE (Rev-response element) and U6 RNA in vitro. Using SELEX (systematic evolution of ligands by exponential enrichment), we have also shown that eIF-5A is capable of binding to RNA in a sequence-specific manner [Xu and Chen (2001) J. Biol. Chem. 276, 2555–2561]. In the present paper, we show that the identification of mRNA species that bind to eIF-5A can be achieved by affinity co-purification and PCR differential display. Using this approach with three sets of anchoring and arbitrary primers, we have found 20 RNA sequences that co-purified specifically with eIF-5A. Five of them contained AAAUGU, the putative eIF-5A-interacting element that we identified previously using the SELEX method. Direct binding of the cloned RNA to eIF-5A could be demonstrated by electrophoretic mobility-shift assay. BLAST analysis revealed that the eIF-5A-interacting RNAs encode proteins such as ribosomal L35a, plasminogen activation inhibitor mRNA-binding protein, NADH dehydrogenase subunit and ADP-ribose pyrophosphatase. Some, however, encode hypothetical proteins. All the cloned RNAs have the potential to form extensive stem-loop structures.

scholarly journals ADAR1 and PACT contribute to efficient translation of transcripts containing HIV-1 trans-activating response (TAR) element

2017 ◽  
Vol 474 (7) ◽  
pp. 1241-1257 ◽  
Author(s):  
Evelyn Chukwurah ◽  
Indhira Handy ◽  
Rekha C. Patel
Keyword(s):  
Viral Proteins ◽  
Antiviral Response ◽  
Cellular Protein ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Protein Activator ◽  
Cellular Antiviral Response ◽  
Innate Antiviral Response ◽  
Hiv 1

Human immunodeficiency virus type 1 (HIV-1) has evolved various measures to counter the host cell's innate antiviral response during the course of infection. Interferon (IFN)-stimulated gene products are produced following HIV-1 infection to limit viral replication, but viral proteins and RNAs counteract their effect. One such mechanism is specifically directed against the IFN-induced Protein Kinase PKR, which is centrally important to the cellular antiviral response. In the presence of viral RNAs, PKR is activated and phosphorylates the translation initiation factor eIF2α. This shuts down the synthesis of both host and viral proteins, allowing the cell to mount an effective antiviral response. PACT (protein activator of PKR) is a cellular protein activator of PKR, primarily functioning to activate PKR in response to cellular stress. Recent studies have indicated that during HIV-1 infection, PACT's normal cellular function is compromised and that PACT is unable to activate PKR. Using various reporter systems and in vitro kinase assays, we establish in this report that interactions between PACT, ADAR1 and HIV-1-encoded Tat protein diminish the activation of PKR in response to HIV-1 infection. Our results highlight an important pathway by which HIV-1 transcripts subvert the host cell's antiviral activities to enhance their translation.

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