The Post-translational Synthesis of Hypusine In eIF5A

eIF5A 中 Hypusine 的翻译后合成

• 批准号: 7593370
• 负责人: MYUNG HEE PARK
• 金额: $ 74.41万
• 依托单位: NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7593370
• 关键词: 2-Oxoglutarate 5-Dioxygenase Procollagen-Lysine; Active Sites; Alanine; Amino Acid Substitution; Amino Acids; Anabolism; Asparagine; Aspartic Acid; Binding; Binding Proteins; Biochemical Reaction; Biological; C-terminal; Cell Proliferation; Charge; Chimeric Proteins; Circular Dichroism; Complement; Dioxygenases; Disease; Elements; Enzymes; Eukaryotic Cell; Eukaryotic Initiation Factors; Exhibits; Glutamic Acid; Glutamine; Glutathione S-Transferase; Goals; Growth; Heating; Helix (Snails); Human; Hydroxylation; Impairment; Individual; Intervention; Iron; Label; Lysine; Mammalian Cell; Mixed Function Oxygenases; Modification; Molecular Mechanisms of Action; Mutation; N-terminal; Numbers; Pathway interactions; Peptides; Plant Resins; Play; Post-Translational Protein Processing; Property; Protein Biosynthesis; Proteins; Reaction; Reporting; Role; Saccharomyces cerevisiae; Sepharose; Sequence Alignment; Side; Site; Specificity; Structure; Testing; Vertebral column; amino group; base; deoxyhypusine; deoxyhypusine monooxygenase; deoxyhypusine synthase; eIF-5A; ear helix; hypusine; inhibitor/antagonist; mutant; novel; polypeptide

In the previous studies, we have identified eIF5A as the only cellular protein that contains an unusual amino acid, hypusine Ne-(4-amino-2-hydroxybutyl)lysine, and established that hypusine biosynthesis occurs by two sequential enzymatic reactions: i) deoxyhypusine synthesis and ii) deoxyhypusine hydroxylation. We demonstrated that hypusine modification is essential for the activity of eIF-5A and for mammalian cell proliferation. We have cloned and characterized the structural and catalytic properties of the two enzymes of the hypusine pathway, deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH), with the aim of developing specific inhibitors. In this reporting period, we have carried out the structure/function studies of DOHH to characterize the basis of the strict specificity of the hypusine modification in one protein. We have also conducted the structure/function studies of eIF5A, and identified the key structural elements of eIF5A critical for its biological activity in supporting growth and protein synthesis. Examination of the DOHH sequence alignment reveals that it is a super helical protein containing eight tandem alpha helical hairpins (HEAT motifs). DOHH is a dyad of symmetrical N-terminal and C-terminal halves, each half consisting of four HEAT repeats, connected by a variable insert. Either the N-terminal half or the C-terminal half was inactive alone and in mixtures, an indication that DOHH does not contain two independent active sites, and that the two symmetrical halves form one active site. We have validated the proposed HEAT-repeat structure by CD spectral analysis. DOHH contains four distinctive, strictly conserved His-Glu motifs. Each the His and Glu residues in these motifs is critical for the DOHH enzymatic activity. We have identified six residues of these motifs (His56, His89, Glu90, His207, His 240 and Glu241) as the iron coordination sites and two residues (Glu57 and Glu208) as those anchoring the protein substrate eIF5A(Dhp). The predicted super helical structure of DOHH is entirely different from the beta jellyroll structure (termed the double stranded beta helix (DSBH)) of the majority of other protein hydroxylases, e.g. the Fe(II)-and 2-oxoacid-dependent dioxygenases, prolyl or lysyl hydroxylases, indicating that DOHH has evolved uniquely and exclusively for eIF5A hydroxylation . Structural requirements of eIF5A as a substrate for DOHH: Importance of the deoxyhypusine side chain The basis of the strict specificity of hypusine modification lies on the specificity of molecular interaction between its biosynthetic enzymes and the protein substrate. In an effort to determine the basis of the specificity of DOHH, we first tested the ability of three eIF5A forms, the precursor, the intermediate and the mature form (eIF5A(Lys), eIF5A(Dhp) and eIF5A(Hpu)) to compete with 3H-labeled eIF5A(Dhp) in the DOHH reaction. eIF5A(Lys) did not inhibit DOHH reaction, even when it was added 100 fold excess over the level of 3H-labeled eIF5A(Dhp), suggesting that the eIF5A precursor does not bind to DOHH. The hypusine-containing mature form, eIF5A(Hpu) showed low inhibition. Thus, the 4-aminobutyl side chain of deoxyhypusine residue seems to be critical for binding to DOHH. In order to determine how much of the eIF5A polypeptide backbone is required for the DOHH reaction, we tested eIF5A(Dhp) fragments with stepwise truncation of 10 amino acids from the N-or C-terminal of eIF5A(Dhp) as substrates. Whereas the eIF5A(Dhp) peptide, aa 1-90 was a good substrate for DHS and DOHH, aa 1-80 and aa 30-90 were poor substrates. Thus, truncation of more than 30 amino acids from the N-terminus, and 74 amino acids from the C-terminus caused a drastic reduction of the substrate activity for the DHS as well as for the DOHH, indicating that a large portion of eIF5A(Dhp) N-terminal domain is required for binding and modification by the two enzymes. Identiffication of amino acid residues of DOHH critical for binding of its protein substrate In order to determine the amino acid residues of DOHH involved in the binding of its substrate, eIF5A(Dhp), we generated 36 mutant enzymes with individual alanine substitution of strictly conserved amino acids and examined their activities, iron contents and abilities to bind the substrate protein. The alpha-helical contents of the mutant enzymes estimated by CD (circular dichroism) analyses were comparable to that of the wild type enzyme, suggesting that there is no gross alteration in the folded structures of these mutant enzymes. Of these 36 mutant enzymes, the eight alanine substitution mutant enzymes of the His-Glu motifs were totally inactive. R26A, G63A, R88A, E93A, R175A, R183A, S202A, G214A, Q215A, M237A and G247A exhibited a large reduction in the activity (by 60-95 %). The iron contents were significantly reduced in six mutant enzymes of the His-Glu motifs, H56A, H89A, E90A, H207A, H240A and E241A, but not in others. The GST fusion proteins of the wild type and mutant enzymes were used to pull down eIF5A(Dhp) using GSH sepharose resin. Whereas the wild type enzyme and a number of mutant enzymes effectively pulled down eIF5A(Dhp), the binding of the substrate protein was undetectable with E57A, and E208A and drastically decreased in G63A and G214A, indicating that the activity loss is due to their inability to bind the protein substrate. The importance of the acidic charges of the two glutamic acid residues (Glu57 and Glu208) was confirmed by substitution of these residues with Gln, Asp and Asn. Only the aspartic acid substitution mutant enzymes (E57D and E208D) were able to bind the substrate protein and were partially active. The glutamine or asparagine substitution enzymes (E57Q, E57N, E208Q and E208N) failed to bind and hydroxylate eIF5A(Dhp). Based on these and above (structural requirement of eIF5A(Dhp)) results, we conclude that the amino groups of the deoxyhypusine side chain of eIF5A(Dhp) is anchored by the two carboxyl side chains of Glu57 and Glu208 at the DOHH active site. This critical ionic interaction between the deoxyhypusine side chain of eIF5A(Dhp) and the acidic groups of Glu57, and Glu208 of the enzyme provides the basis of strict specificity of DOHH reaction. Mutational analysis of eIF5A: Identification of amino acid residues critical for the biological activity of eIF5A and the hypusine modification. To investigate the features of eIF5A required for its activity, we generated 49 mutations in the human eIF5A-1, with a single amino acid substitution at each of the highly conserved residues or with N-or C-terminal truncations, and tested the mutant proteins in complementing the growth of a S. cerevisiae eIF5A null strain. Growth supporting activity was abolished in only a few mutant eIF5As, those with substitutions at or near the hypusine modification site (K47D, G49A, K50A, K50D, K50I, K50R, G52A and K55A) or those with truncation of 21 amino acids from either the N-or C-terminus. For the Lys50 substitution proteins and G52A and K55A, the inactivity is largely due to the lack or impairment of deoxyhypusine modification. In contrast, K47D and G49A were effective substrates for deoxyhypusine synthase, yet failed to support growth, suggesting critical importance of Lys47 and Gly49 in eIF5A biological activity, possibly in its interaction with downstream effector(s). These results demomstrate that both N- and C-terminal domains of eIF5A are required for its biological activity and that the hypusine residue and the hypusine containing loop play a critical role in the biological action of eIF5A possibly in its interaction with its downstream effectors.

在之前的研究中，我们已经确定了eIF5A是唯一含有一种不寻常的氨基酸- hypusine Ne-（4-氨基-2-羟基丁基）赖氨酸的细胞蛋白，并确定了hypusine的生物合成发生在两个连续的酶促反应中：i)脱氧hypusine合成和ii)脱氧hypusine羟基化。我们证明了hypusine修饰对于eIF-5A的活性和哺乳动物细胞增殖是必不可少的。我们克隆并表征了hypusine途径的两个酶，脱氧hypusine合成酶（DHS）和脱氧hypusine羟化酶（DOHH）的结构和催化性能，目的是开发特异性抑制剂。在本报告期间，我们进行了DOHH的结构/功能研究，以表征一种蛋白质中hypusine修饰的严格特异性的基础。我们还进行了eIF5A的结构/功能研究，并确定了eIF5A在支持生长和蛋白质合成的生物活性中至关重要的关键结构元件。