Mutational analyses of human eIF5A-1--identification of amino acid residues critical for eIF5A activity and hypusine modification

The eukaryotic translation initiation factor 5A (eIF5A) is the only protein that contains hypusine [Nepsilon-(4-amino-2-hydroxybutyl)lysine], which is required for its activity. Hypusine is formed by post-translational modification of one specific lysine (Lys50 for human eIF5A) by deoxyhypusine synthase and deoxyhypusine hydroxylase. To investigate the features of eIF5A required for its activity, we generated 49 mutations in human eIF5A-1, with a single amino acid substitution at the highly conserved residues or with N-terminal or C-terminal truncations, and tested mutant proteins in complementing the growth of a Saccharomyces cerevisiae eIF5A null strain. Growth-supporting activity was abolished in only a few mutant eIF5As (K47D, G49A, K50A, K50D, K50I, K50R, G52A and K55A), with substitutions at or near the hypusine modification site or with truncation of 21 amino acids from either the N-terminus or C-terminus. The inactivity of the Lys50 substitution proteins is obviously due to lack of deoxyhypusine modification. In contrast, K47D and G49A were effective substrates for deoxyhypusine synthase, yet failed to support growth, suggesting critical roles of Lys47 and Gly49 in eIF5A activity, possibly in its interaction with effector(s). By use of a UBHY-R strain harboring genetically engineered unstable eIF5A, we present evidence for the primary function of eIF5A in protein synthesis. When selected eIF5A mutant proteins were tested for their activity in protein synthesis, a close correlation was observed between their ability to enhance protein synthesis and growth, lending further support for a central role of eIF5A in translation.     

Deoxyhypusine Modification of Eukaryotic Translation Initiation Factor 5A (eIF5A) Is Essential for Trypanosoma brucei Growth and for Expression of Polyprolyl-containing Proteins

The eukaryotic protozoan parasite Trypanosoma brucei is the causative agent of human African trypanosomiasis. Polyamine biosynthesis is essential in T. brucei, and the polyamine spermidine is required for synthesis of a novel cofactor called trypanothione and for deoxyhypusine modification of eukaryotic translation initiation factor 5A (eIF5A). eIF5A promotes translation of proteins containing polyprolyl tracts in mammals and yeast. To evaluate the function of eIF5A in T. brucei, we used RNA interference (RNAi) to knock down eIF5A levels and found that it is essential for T. brucei growth. The RNAi-induced growth defect was complemented by expression of wild-type human eIF5A but not by a Lys-50 mutant that blocks modification by deoxyhypusine. Bioinformatics analysis showed that 15% of the T. brucei proteome contains 3 or more consecutive prolines and that actin-related proteins and cysteine proteases were highly enriched in the group. Steady-state protein levels of representative proteins containing 9 consecutive prolines that are involved in actin assembly (formin and CAP/Srv2p) were significantly reduced by knockdown of eIF5A. Several T. brucei polyprolyl proteins are involved in flagellar assembly. Knockdown of TbeIF5A led to abnormal cell morphologies and detached flagella, suggesting that eIF5A is important for translation of proteins needed for these processes. Potential specialized functions for eIF5A in T. brucei in translation of variable surface glycoproteins were also uncovered. Inhibitors of deoxyhypusination would be expected to cause a pleomorphic effect on multiple cell processes, suggesting that deoxyhypusine/hypusine biosynthesis could be a promising drug target in not just T. brucei but in other eukaryotic pathogens.     

The N-terminal region of eukaryotic translation initiation factor 5A signals to nuclear localization of the protein

The eukaryotic translation initiation factor 5A (eIF5A) is a ubiquitous protein of eukaryotic and archaeal organisms which undergoes hypusination, a unique post-translational modification. We have generated a polyclonal antibody against murine eIF5A, which in immunocytochemical assays in B16-F10 cells revealed that the endogenous protein is preferentially localized to the nuclear region. We therefore analyzed possible structural features present in eIF5A proteins that could be responsible for that characteristic. Multiple sequence alignment analysis of eIF5A proteins from different eukaryotic and archaeal organisms showed that the former sequences have an extended N-terminal segment. We have then performed in silico prediction analyses and constructed different truncated forms of murine eIF5A to verify any possible role that the N-terminal extension might have in determining the subcellular localization of the eIF5A in eukaryotic organisms. Our results indicate that the N-terminal extension of the eukaryotic eIF5A contributes in signaling this protein to nuclear localization, despite of bearing no structural similarity with classical nuclear localization signals.     

Acetylation regulates subcellular localization of eukaryotic translation initiation factor 5A (eIF5A)

Eukaryotic translation initiation factor 5A (eIF5A) is a protein subject to hypusination, which is essential for its function. eIF5A is also acetylated, but the role of that modification is unknown. Here, we report that acetylation regulates the subcellular localization of eIF5A. We identified PCAF as the major cellular acetyltransferase of eIF5A, and HDAC6 and SIRT2 as its major deacetylases. Inhibition of the deacetylases or impaired hypusination increased acetylation of eIF5A, leading to nuclear accumulation. As eIF5A is constitutively hypusinated under physiological conditions, we suggest that reversible acetylation plays a major role in controlling the subcellular localization of eIF5A.     

真核翻译起始因子5A在蛋白质合成中的功能及机制

在蛋白质合成过程中,除核糖体、氨酰 tRNA和mRNA外,还有多种翻译因子参与其中。真核翻译起始因子5A（eukaryotic translation initiation factor 5A, eIF5A）是维持细胞活性必不可少的翻译因子,在进化上高度保守。eIF5A是真核细胞中唯一含有羟腐胺赖氨酸（hypusine）的蛋白质,该翻译后修饰对eIF5A的活性至关重要。1978年,人们首次鉴定出eIF5A,认为它在翻译起始阶段促进第1个肽键的形成。直到2013年才证实它主要在翻译延伸阶段调控含多聚脯氨酸基序蛋白质的翻译。在经过四十多年研究后,人们对eIF5A的功能有了新的认识。近期基于核糖体图谱数据的分析表明,eIF5A能够缓解翻译延伸过程中核糖体在多种基序处的停滞,并不局限于多聚脯氨酸基序,并且它还能够通过促进肽链的释放增强翻译终止。此外,eIF5A还可以通过调控某些蛋白质的翻译,间接影响细胞内的各种生命活动。本文综述了eIF5A的多种翻译后修饰、在蛋白质合成和细胞自噬过程中的调控作用以及与人类疾病的关系,并与细菌及古细菌中的同源蛋白质进行了比较,探讨了该因子在进化中的保守性,以期为相关领域的研究提供一定的理论基础。     

Inactivation of eukaryotic initiation factor 5A (eIF5A) by specific acetylation of its hypusine residue by spermidine/spermine acetyltransferase 1 (SSAT1)

eIF5A (eukaryotic translation initiation factor 5A) is the only cellular protein containing hypusine [N?-(4-amino-2-hydroxybutyl)lysine]. eIF5A is activated by the post-translational synthesis of hypusine and the hypusine modification is essential for cell proliferation. In the present study, we report selective acetylation of the hypusine and/or deoxyhypusine residue of eIF5A by a key polyamine catabolic enzyme SSAT1 (spermidine/spermine-N1-acetyltransferase 1). This enzyme normally catalyses the N1-acetylation of spermine and spermidine to form acetyl-derivatives, which in turn are degraded to lower polyamines. Although SSAT1 has been reported to exert other effects in cells by its interaction with other cellular proteins, eIF5A is the first target protein specifically acetylated by SSAT1. Hypusine or deoxyhypusine, as the free amino acid, does not act as a substrate for SSAT1, suggesting a macromolecular interaction between eIF5A and SSAT1. Indeed, the binding of eIF5A and SSAT1 was confirmed by pull-down assays. The effect of the acetylation of hypusine on eIF5A activity was assessed by comparison of acetylated with non-acetylated bovine testis eIF5A in the methionyl-puromycin synthesis assay. The loss of eIF5A activity by this SSAT1-mediated acetylation confirms the strict structural requirement for the hypusine side chain and suggests a possible regulation of eIF5A by hypusine acetylation/deacetylation.     

The hypusine-containing translation factor eIF5A

Abstract

In addition to the small and large ribosomal subunits, aminoacyl-tRNAs, and an mRNA, cellular protein synthesis is dependent on translation factors. The eukaryotic translation initiation factor 5A (eIF5A) and its bacterial ortholog elongation factor P (EF-P) were initially characterized based on their ability to stimulate methionyl-puromycin (Met-Pmn) synthesis, a model assay for protein synthesis; however, the function of these factors in cellular protein synthesis has been difficult to resolve. Interestingly, a conserved lysine residue in eIF5A is post-translationally modified to hypusine and the corresponding lysine residue in EF-P from at least some bacteria is modified by the addition of a β-lysine moiety. In this review, we provide a summary of recent data that have identified a novel role for the translation factor eIF5A and its hypusine modification in the elongation phase of protein synthesis and more specifically in stimulating the production of proteins containing runs of consecutive proline residues.     

Post-translational modification by β-lysylation is required for activity of Escherichia coli elongation factor P (EF-P)

Bacterial elongation factor P (EF-P) is the ortholog of archaeal and eukaryotic initiation factor 5A (eIF5A). EF-P shares sequence homology and crystal structure with eIF5A, but unlike eIF5A, EF-P does not undergo hypusine modification. Recently, two bacterial genes, yjeA and yjeK, encoding truncated homologs of class II lysyl-tRNA synthetase and of lysine-2,3-aminomutase, respectively, have been implicated in the modification of EF-P to convert a specific lysine to a hypothetical β-lysyl-lysine. Here we present biochemical evidence for β-lysyl-lysine modification in Escherichia coli EF-P and for its role in EF-P activity by characterizing native and recombinant EF-P proteins for their modification status and activity in vitro. Mass spectrometric analyses confirmed the lysyl modification at lysine 34 in native and recombinant EF-P proteins. The β-lysyl-lysine isopeptide was identified in the exhaustive Pronase digests of native EF-P and recombinant EF-P isolated from E. coli coexpressing EF-P, YjeA, and YjeK but not in the digests of proteins derived from the vectors encoding EF-P alone or EF-P together with YjeA, indicating that both enzymes, YjeA and YjeK, are required for β-lysylation of EF-P. Endogenous EF-P as well as the recombinant EF-P preparation containing β-lysyl-EF-P stimulated N-formyl-methionyl-puromycin synthesis ～4-fold over the preparations containing unmodified EF-P and/or α-lysyl-EF-P. The mutant lacking the modification site lysine (K34A) was inactive. This is the first report of biochemical evidence for the β-lysylation of EF-P in vivo and the requirement for this modification for the activity of EF-P.     

eIF5A interacts functionally with eEF2

eIF5A is highly conserved from archaea to mammals, essential for cell viability and the only protein known to contain the essential amino acid residue hypusine, generated by a unique posttranslational modification. eIF5A was originally identified as a translation initiation factor due to its ability to stimulate the formation of the first peptide bond. However, recent studies have shown that depletion of eIF5A causes a significant decrease in polysome run-off and an increase in the ribosome transit time, suggesting that eIF5A is actually involved in the elongation step of protein synthesis. We have previously shown that the depletion mutant tif51A-3 (eIF5A(C39Y/G118D)) shows a sicker phenotype when combined with the dominant negative mutant eft2 ( H699K ) of the elongation factor eEF2. In this study, we used the eIF5A(K56A) mutant to further investigate the relationship between eIF5A and eEF2. The eIF5A(K56A) mutant is temperature sensitive and has a defect in protein synthesis, but instead of causing depletion of the eIF5A protein, this mutant has a defect in hypusine modification. Like the mutant tif51A-3, the eIF5A(K56A) mutant is synthetic sick with the mutant eft2 ( H699K ) of eEF2. High-copy eEF2 not only improves cell growth of the eIF5A(K56A) mutant, but also corrects its increased cell size defect. Moreover, eEF2 suppression of the eIF5A(K56A) mutant is correlated with the improvement of total protein synthesis and with the increased resistance to the protein synthesis inhibitor hygromycin B. Finally, the polysome profile defect of the eIF5A(K56A) mutant is largely corrected by high-copy eEF2. Therefore, these results demonstrate that eIF5A is closely related to eEF2 function during translation elongation.     

Posttranslational synthesis of hypusine: evolutionary progression and specificity of the hypusine modification

Summary. A naturally occurring unusual amino acid, hypusine [N ^ɛ-(4-amino-2-hydroxybutyl)-lysine] is a component of a single cellular protein, eukaryotic translation initiation factor 5A (eIF5A). It is a modified lysine with structural contribution from the polyamine spermidine. Hypusine is formed in a novel posttranslational modification that involves two enzymes, deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH). eIF5A and deoxyhypusine/hypusine modification are essential for growth of eukaryotic cells. The hypusine synthetic pathway has evolved in eukaryotes and eIF5A, DHS and DOHH are highly conserved, suggesting maintenance of a fundamental cellular function of eIF5A through evolution. The unique feature of the hypusine modification is the strict specificity of the enzymes toward its substrate protein, eIF5A. Moreover, DHS exhibits a narrow specificity toward spermidine. In view of the extraordinary specificity and the requirement for hypusine-containing eIF5A for mammalian cell proliferation, eIF5A and the hypusine biosynthetic enzymes present new potential targets for intervention in aberrant cell proliferation.     

Tandem affinity purification revealed the hypusine-dependent binding of eukaryotic initiation factor 5A to the translating 80S ribosomal complex

Eukaryotic initiation factor 5A (eIF5A) is the only protein in nature that contains hypusine, an unusual amino acid formed post-translationally in two steps by deoxyhypusine synthase and deoxyhypusine hydroxylase. Genes encoding eIF5A or deoxyhypusine synthase are essential for cell survival and proliferation. To determine the physiological function of eIF5A, we have employed the tandem affinity purification (TAP) method and mass spectrometry to search for and identify the potential eIF5A-interacting proteins. The TAP-tag was fused in-frame to chromosomal TIF51A gene and eIF5A-TAP fusion protein expressed at its natural level was used as the bait to fish out its interacting partners. At salt concentrations of 150 mM, deoxyhypusine synthase was the only protein bound to eIF5A. As salt concentrations were lowered to 125 mM or less, eIF5A interacted with a set of proteins, which were identified as the components of the 80S ribosome complex. The eIF5A-ribosome interaction was sensitive to RNase and EDTA treatments, indicating the requirement of RNA and the joining of 40S and 60S ribosomal subunits for the interaction. Importantly, a single mutation of hypusine to arginine completely abolished the eIF5A-ribosome interaction. Sucrose gradient sedimentation analysis of log versus stationary phase cells and eIF3 mutant strain showed that the endogenous eIF5A co-sedimented with the actively translating 80S ribosomes and polyribosomes in an RNase- and EDTA-sensitive manner. Our study demonstrates for the first time that eIF5A interacts in a hypusine-dependent manner with a molecular complex rather than a single protein, suggesting that the essential function of eIF5A is mostly likely mediated through its interaction with the actively translating ribosomes.     

Structural modeling and mutational analysis of yeast eukaryotic translation initiation factor 5A reveal new critical residues and reinforce its involvement in protein synthesis

Eukaryotic translation initiation factor 5A (eIF5A) is a protein that is highly conserved and essential for cell viability. This factor is the only protein known to contain the unique and essential amino acid residue hypusine. This work focused on the structural and functional characterization of Saccharomyces cerevisiae eIF5A. The tertiary structure of yeast eIF5A was modeled based on the structure of its Leishmania mexicana homologue and this model was used to predict the structural localization of new site-directed and randomly generated mutations. Most of the 40 new mutants exhibited phenotypes that resulted from eIF-5A protein-folding defects. Our data provided evidence that the C-terminal alpha-helix present in yeast eIF5A is an essential structural element, whereas the eIF5A N-terminal 10 amino acid extension not present in archaeal eIF5A homologs, is not. Moreover, the mutants containing substitutions at or in the vicinity of the hypusine modification site displayed nonviable or temperature-sensitive phenotypes and were defective in hypusine modification. Interestingly, two of the temperature-sensitive strains produced stable mutant eIF5A proteins--eIF5A(K56A) and eIF5A(Q22H,L93F)--and showed defects in protein synthesis at the restrictive temperature. Our data revealed important structural features of eIF5A that are required for its vital role in cell viability and underscored an essential function of eIF5A in the translation step of gene expression.     

Eukaryotic translation initiation factor 5A induces apoptosis in colon cancer cells and associates with the nucleus in response to tumour necrosis factor alpha signalling

Eukaryotic translation initiation factor 5A (eIF5A) is thought to function as a nucleocytoplasmic shuttle protein. There are reports of its involvement in cell proliferation, and more recently it has also been implicated in the regulation of apoptosis. In the present study, we examined the effects of eIF5A over-expression on apoptosis and of siRNA-mediated suppression of eIF5A on expression of the tumour suppressor protein, p53. Over-expression of either eIF5A or a mutant of eIF5A incapable of being hypusinated was found to induce apoptosis in colon carcinoma cells. Our results also indicate that eIF5A is required for expression of p53 following the induction of apoptosis by treatment with Actinomycin D. Depiction of eIF5A localization by indirect immunofluorescence has indicated, for the first time, that the protein is rapidly translocated from the cytoplasm to the nucleus by death receptor activation or following treatment with Actinomycin D. These findings collectively indicate that unhypusinated eIF5A may have pro-apoptotic functions and that eIF5A is rapidly translocated to the nucleus following the induction of apoptotic cell death.     

Rapid depletion of mutant eukaryotic initiation factor 5A at restrictive temperature reveals connections to actin cytoskeleton and cell cycle progression

Eukaryotic initiation factor 5A (eIF5A) is the only protein in nature that contains hypusine, an unusual amino acid derived from the modification of lysine by spermidine. Two genes, TIF51A and TIF51B, encode eIF5A in the yeast Saccharomyces cerevisiae. In an effort to understand the structure–function relationship of eIF5A, we have generated yeast mutants by introducing plasmid-borne tif51A into a double null strain where both TIF51A and TIF51B have been disrupted. One of the mutants, tsL102A strain (tif51A L102A tif51aΔ tif51bΔ) exhibits a strong temperature-sensitive growth phenotype. At the restrictive temperature, tsL102A strain also exhibits a cell shape change, a lack of volume change in response to temperature increase and becomes more sensitive to ethanol, a hallmark of defects in the PKC/WSC cell wall integrity pathway. In addition, a striking change in actin dynamics and a complete cell cycle arrest at G1 phase occur in tsL102A cells at restrictive temperature. The temperature-sensitivity of tsL102A strain is due to a rapid loss of mutant eIF5A with the half-life reduced from 6 h at permissive temperature to 20 min at restrictive temperature. Phenylmethyl sulfonylfluoride (PMSF), an irreversible inhibitor of serine protease, inhibited the degradation of mutant eIF5A and suppressed the temperature-sensitive growth arrest. Sorbitol, an osmotic stabilizer that complement defects in PKC/WSC pathways, stabilizes the mutant eIF5A and suppresses all the observed temperature-sensitive phenotypes.     

Molecular modeling of the human eukaryotic translation initiation factor 5A (eIF5A) based on spectroscopic and computational analyses

The eukaryotic translation initiation factor 5A (eIF5A) is a protein ubiquitously present in archaea and eukarya, which undergoes a unique two-step post-translational modification called hypusination. Several studies have shown that hypusination is essential for a variety of functional roles for eIF5A, including cell proliferation and synthesis of proteins involved in cell cycle control. Up to now neither a totally selective inhibitor of hypusination nor an inhibitor capable of directly binding to eIF5A has been reported in the literature. The discovery of such an inhibitor might be achieved by computer-aided drug design based on the 3D structure of the human eIF5A. In this study, we present a molecular model for the human eIF5A protein based on the crystal structure of the eIF5A from Leishmania brasiliensis, and compare the modeled conformation of the loop bearing the hypusination site with circular dichroism data obtained with a synthetic peptide of this loop. Furthermore, analysis of amino acid variability between different human eIF5A isoforms revealed peculiar structural characteristics that are of functional relevance.     

Biological Relevance and Therapeutic Potential of the Hypusine Modification System

Hypusine modification of the eukaryotic initiation factor 5A (eIF-5A) is emerging as a crucial regulator in cancer, infections, and inflammation. Although its contribution in translational regulation of proline repeat-rich proteins has been sufficiently demonstrated, its biological role in higher eukaryotes remains poorly understood. To establish the hypusine modification system as a novel platform for therapeutic strategies, we aimed to investigate its functional relevance in mammals by generating and using a range of new knock-out mouse models for the hypusine-modifying enzymes deoxyhypusine synthase and deoxyhypusine hydroxylase as well as for the cancer-related isoform eIF-5A2. We discovered that homozygous depletion of deoxyhypusine synthase and/or deoxyhypusine hydroxylase causes lethality in adult mice with different penetrance compared with haploinsufficiency. Network-based bioinformatic analysis of proline repeat-rich proteins, which are putative eIF-5A targets, revealed that these proteins are organized in highly connected protein-protein interaction networks. Hypusine-dependent translational control of essential proteins (hubs) and protein complexes inside these networks might explain the lethal phenotype observed after deletion of hypusine-modifying enzymes. Remarkably, our results also demonstrate that the cancer-associated isoform eIF-5A2 is dispensable for normal development and viability. Together, our results provide the first genetic evidence that the hypusine modification in eIF-5A is crucial for homeostasis in mammals. Moreover, these findings highlight functional diversity of the hypusine system compared with lower eukaryotes and indicate eIF-5A2 as a valuable and safe target for therapeutic intervention in cancer.     

Effect of N 1-guanyl-1,7-diaminoheptane,an inhibitor of deoxyhypusine synthase,on endothelial cell growth,differentiation and apoptosis

An unusual amino acid, hypusine [N -(4-amino-2-hydroxybutyl)lysine], is formed post-translationally in a single cellular protein, the eukaryotic translation initiation factor 5A (eIF5A) by deoxyhypusine synthase and deoxyhypusine hydroxylase. Although eIF5A and its hypusine modification are essential for eukaryotic cell viability, the true physiological function of eIF5A is yet unknown. We have examined the effects of N ¹-guanyl-1,7-diaminoheptane (GC7), a potent inhibitor of deoxyhypusine synthase, on endothelial cell proliferation, differentiation and apoptosis. Upon treatment of human umbilical vein endothelial cells (HUVEC) with GC7, dose-dependent inhibition of hypusine formation and cellular proliferation was observed. GC7 at 10 M caused almost complete inhibition of cellular hypusine synthesis and led to cytostasis of HUVEC. Pretreatment of HUVEC with GC7 up to 50 M for 4 days had little effect on the attachment and differentiation of these cells on Matri-gel and did not cause induction of apoptosis. Instead, the GC7 pretreatment (96 h at 5–50 M) elicited protective effects against apoptotic death of HUVEC induced by serum starvation. These results suggest that eIF-5A may be involved in expression of proteins essential for apoptosis of endothelial cells as well as those for cellular proliferation.     

eIF5A has a function in the elongation step of translation in yeast

The putative translation factor eIF5A is essential for cell viability and is highly conserved throughout evolution. Here, we describe genetic interactions between an eIF5A mutant and a translation initiation mutant (eIF4E) or a translation elongation mutant (eEF2). Polysome profile analysis of single and double mutants revealed that mutation in eIF5A reduces polysome run-off, contrarily to translation initiation mutants. Moreover, the polysome profile of an eIF5A mutant alone is very similar to that of a translation elongation mutant. Furthermore, depletion of eIF5A causes a significant decrease in total protein synthesis and an increase of the average ribosome transit time. Finally, we demonstrate that the formation of P bodies is inhibited in an eIF5A mutant, similarly to the effect of the translation elongation inhibitor cycloheximide. Taken together, these results not only reinforce a role for eIF5A in translation but also strongly support a function for eIF5A in the elongation step of protein synthesis.     

Pumpkin eIF5A isoforms interact with components of the translational machinery in the cucurbit sieve tube system

In yeast, eIF5A, in combination with eEF2, functions at the translation step, during the protein elongation cycle. This result is of significance with respect to functioning of the enucleate sieve tube system, as eIF5A was recently detected in Cucurbita maxima (pumpkin) phloem sap. In the present study, we further characterized four CmeIF5A isoforms, encoding three proteins, all of which were present in the phloem sap. Although hypusination of CmeIF5A was not necessary for entry into the sieve elements, this unique post‐translational modification was necessary for RNA binding. The two enzymes required for hypusination were detected in pumpkin phloem sap, where presumably this modification takes place. A combination of gel‐filtration chromatography and protein overlay assays demonstrated that, as in yeast, CmeIF5A interacts with phloem proteins, like eEF2, known to be involved in protein synthesis. These findings are discussed in terms of a potential role for eIF5A in regulating protein synthesis within the enucleate sieve tube system of the angiosperms.