The Post-translational Synthesis of Hypusine In eIF5A

eIF5A 中 Hypusine 的翻译后合成

• 批准号: 7318819
• 负责人: MYUNG HEE PARK
• 金额: --
• 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/7318819
• 关键词: Post; translational; Synthesis; Hypusine; eIF5A

In 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 purified deoxyhypusine synthase (DHS), cloned its cDNA and extensively characterized the structural and catalytic properties of this first step enzyme. We developed potent inhibitors of DHS that exert strong antiproliferative effects in mammalian cells, including a panel of human cancer cell lines. In 2005, we have succeeded in the molecular cloning and expression of the second step enzyme, deoxyhypusine hydroxylase (DOHH). In the past reporting period, we have made a major progress in the characterization of the structure and mechanism of DOHH. The availability of the DOHH clone and protein has enabled us to overexpress fully modified eIF5A in mammalian cells and to make new approaches in the eIF5A functional studies. Cloning, Expression and purification of the recombinant yeast and human DOHH. We over-expressed human and S. cerevisiae DOHH as GST-fusion proteins in E. coli BL23(DE3) cells using the bacterial expression vector, pGEX-4T-3. The GST-fusion enzymes were purified on GSH-Sepharose and free DOHH enzymes were purified by thrombin cleavage of the GSH-resin bound GST-DOHH. The human enzyme is a smaller protein (32 kDa protein with 302 amino acids) than the S. cerevisiae enzyme (36 kDa protein with 325 amino acids). The purified yeast and human enzymes showed comparable activities in the conversion of the deoxyhypusine-containing eIF5A to the mature hypusine-containing eIF5A. These findings confirm that the cloned yeast and human genes indeed encode DOHH enzymes. DOHH is a novel HEAT-repeat-containing metalloenzyme with a structure distinct from other protein hydroxylases Examination of the DOHH sequence alignment reveals that it is a super helical protein containing eight tandem alpha helical hairpins (HEAT repeats). DOHH is a dyad of symmetrical N-terminal and C-terminal halves, each half consisting of four HEAT repeats, connected by a variable insert. The N-terminal half or 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. DOHH contains four distinctive, strictly conserved HE (histidine-glutamate) motifs, potential metal coordination site. The DOHH structural model predicts that metal-chelating sites lie in the interior of the concave structure formed by two toroid arms. In order to validate the proposed HEAT repeat structure, CD spectral analysis was performed for the purified human DOHH. It revealed 77 % helical content, close to the value (76-78%) calculated from the predicted structure. 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. Site-directed mutagenesis of DOHH and identification of amino acid residues critical for iron binding and catalysis In order to validate the proposed metal binding sites of DOHH, we have generated mutant enzymes with alanine substitutions of the conserved His and Glu residues. Eight single mutants, H56A, E57A, H89A, E90A, H207A, E208A, H240A, and E241A were totally devoid of DOHH activity, indicating their critical role in DOHH catalysis. Of these, six (H56A, H89A, E90A, H207A, H240A and E241A) had markedly reduced iron content, indicating that the six conserved His or Glu residues are involved in the coordination of iron at the acctive site of DOHH. Consistent with the total lack of activity of N-terminal or C-terminal half of DOHH, this finding indicates that both the iron binding and catalysis are coordinated at one active center composed of the four His-Glu motifs. The two conserved residues of His-Glu motifs, E57 and E208 that are not important for iron binding, are required for the binding of substrate protein. DOHH is a Fe(II)-dependent enzyme with a di-iron active center: a reaction mechanism distinct from other protein hydroxylases. DOHH holoenzyme was separated from the metal-free, inactive apoenzyme by gel filtration and by native gel electrophoresis, since the hydrodynamic size of the apoenzyme is larger than the iron-containing holoenzyme. The stoichiometry of iron in the holoenzyme fraction was determined to be close to two mol of iron per mol of DOHH. Of various metals tested only Fe(II), but not Fe(III), stimulated the activity of the apoenzyme, indicating that Fe(II) is the catalytic metal for DOHH. The iron to DOHH stoichiometry and dependence of the iron binding and DOHH activity on each of the four conserved His-Glu motifs suggest that DOHH monomer has one dinuclear iron-containing active center, like other non-heme iron-dependent monooxygenases, such as ribonucleotide reductase and methane momooxygenase. Furthermore, addition of 2-oxoglutarate did not enhance DOHH activity of the human recombinant enzyme suggesting that its reaction mechanism is distinct from that of other 2-oxoacid and Fe(II)-dependent dioxygenases, such as prolyl 4-hydroxylase or lysyl hydroxylases. Co-transfection with vectors encoding eIF5A, DHS and DOHH is required for overproduction of hypusine-containing, active eIF5A in mammalian cells. In order to determine whether the cloned human DOHH gene is functional in cultured human cells, we expressed the human DOHH ORF in combination with human eIF5A-1 and DHS ORFs using mammalian expression vectors. 293T cells were transfected with p3XFLAG-CMV-7.1/heIF5A-1 alone or in combination with pCEFL/hDHS and pCEFL/hDOHH. Western blotting with eIF5A-1 antibody showed over-expression of the FLAG-eIF5A-1 in all samples transfected with p3XFLAG-CMV-7.1/heIF5A-1. However, no labeling of the FLAG-eIF5A-1 was detectable in cells transfected with FLAG-eIF5A-1 vector alone, indicating that in this case, the FLAG-eIF5A-1 protein largely exists as the unmodified precursor. Only upon co-transfection with the modification enzyme DHS, enhanced labeling of FLAG-eIF5A-1 was manifest. The labeled component of Flag-eIF5A-1in this case was deoxyhypusine, but not hypusine, indicating that DOHH was limitting. Labeling of Flag-eIF5A-1 as the hypusine form was observed only after co-transfection of FLAG-eIF5A-1 vector with both DHS and DOHH vectors. These data demonstrate that the cloned human DOHH gene expresses functional DOHH activity in mammalian cells and that co-expression of all three proteins, eIF5A, DHS and DOHH is required for overproduction of fully modified eIF5A in mammalian cells. Differential expression of eIF5A-1 and eIF5A-2 in human cancer cells Of the two human eIF5A genes reported, normally only one isoform (eIF5A-1) is expressed in most human cell lines. eIF5A-1 and eIF5A-2 genes are differentially expressed in mammalian cells. Whereas eIF5A-1 mRNA and protein are constitutively expressed in all human cells, eIF5A-2 mRNAs (0.6-5.5 kb) are expressed in a tissue-specific manner. eIF5A-2 protein was detectable only in the colorectal and ovarian cancer-derived cell lines SW-480 and UACC-1598, which showed high over-expression of eIF5A-2 mRNAs. The multiple forms of eIF5A-2 mRNA, with varying lengths of the 3'-UTR were identified as products of one gene resulting from the use of different polyadenylation signals (AATAAA) in the 3'-UTR.

在之前的研究中，我们已经确定 eIF5A 是唯一含有一种不寻常的氨基酸——山马尿苷 [Ne-(4-氨基-2-羟丁基)赖氨酸] 的细胞蛋白，并确定山马尿苷生物合成是通过两个连续的酶促反应发生的：i) 脱氧马尿苷合成和 ii) 脱氧马尿苷羟基化。我们证明，hypusine 修饰对于 eIF-5A 的活性和哺乳动物细胞增殖至关重要。我们纯化了脱氧马尿苷合酶 (DHS)，克隆了其 cDNA，并广泛表征了该第一步酶的结构和催化特性。我们开发了 DHS 的强效抑制剂，对哺乳动物细胞（包括一组人类癌细胞系）发挥强大的抗增殖作用。 2005年，我们成功实现了第二步酶脱氧马尿碱羟化酶（DOHH）的分子克隆和表达。在过去的报告期内，我们在 DOHH 结构和机制的表征方面取得了重大进展。 DOHH 克隆和蛋白质的可用性使我们能够在哺乳动物细胞中过表达完全修饰的 eIF5A，并在 eIF5A 功能研究中采用新方法。 重组酵母和人 DOHH 的克隆、表达和纯化。 我们使用细菌表达载体 pGEX-4T-3 在大肠杆菌 BL23(DE3) 细胞中过表达人类和酿酒酵母 DOHH 作为 GST 融合蛋白。 GST-融合酶在GSH-Sepharose上纯化，游离DOHH酶通过凝血酶切割GSH-树脂结合的GST-DOHH来纯化。人类酶是比酿酒酵母酶（36 kDa 蛋白质，325 个氨基酸）更小的蛋白质（32 kDa 蛋白质，302 个氨基酸）。纯化的酵母和人酶在将含有脱氧马尿苷的 eIF5A 转化为成熟的含有马尿苷的 eIF5A 方面表现出相当的活性。这些发现证实克隆的酵母和人类基因确实编码 DOHH 酶。 DOHH 是一种新型的含有 HEAT 重复序列的金属酶，其结构不同于其他蛋白质羟化酶 对 DOHH 序列比对的检查表明，它是一种超螺旋蛋白，含有八个串联 α 螺旋发夹（HEAT 重复序列）。 DOHH 是对称的 N 端和 C 端半部的二元组，每半部由四个 HEAT 重复组成，通过可变插入片段连接。 N 端半部或 C 端半部单独或在混合物中均无活性，这表明 DOHH 不包含两个独立的活性位点，并且两个对称的半部形成一个活性位点。 DOHH 包含四个独特的、严格保守的 HE（组氨酸-谷氨酸）基序，即潜在的金属配位位点。 DOHH 结构模型预测金属螯合位点位于由两个环形臂形成的凹结构的内部。为了验证所提出的 HEAT 重复结构，对纯化的人 DOHH 进行了 CD 光谱分析。结果显示螺旋含量为 77%，接近根据预测结构计算出的值 (76-78%)。 DOHH 的预测超螺旋结构与大多数其他蛋白质羟化酶的 β 果冻卷结构（称为双链 β 螺旋 (DSBH)）完全不同，例如Fe(II)-和 2-含氧酸依赖性双加氧酶、脯氨酰或赖氨酰羟化酶。 DOHH 的定点诱变以及对铁结合和催化至关重要的氨基酸残基的鉴定 为了验证所提出的 DOHH 金属结合位点，我们生成了用丙氨酸取代保守的 His 和 Glu 残基的突变酶。八个单突变体 H56A、E57A、H89A、E90A、H207A、E208A、H240A 和 E241A 完全缺乏 DOHH 活性，表明它们在 DOHH 催化中发挥关键作用。其中，六个（H56A、H89A、E90A、H207A、H240A 和 E241A）铁含量显着降低，表明六个保守的 His 或 Glu 残基参与 DOHH 活性位点铁的配位。与 DOHH 的 N 端或 C 端一半完全缺乏活性一致，这一发现表明铁结合和催化作用在由四个 His-Glu 基序组成的一个活性中心协调。 His-Glu 基序的两个保守残基 E57 和 E208 对于铁结合并不重要，但对于底物蛋白的结合是必需的。 DOHH 是一种 Fe(II) 依赖性酶，具有双铁活性中心：其反应机制不同于其他蛋白质羟化酶。 由于脱辅基酶的流体动力学尺寸大于含铁全酶，因此通过凝胶过滤和非变性凝胶电泳将DOHH全酶与无金属、无活性的脱辅基酶分离。全酶部分中铁的化学计量被确定为接近每摩尔 DOHH 两摩尔铁。在所测试的各种金属中，只有 Fe(II) 而不是 Fe(III) 刺激脱辅基酶的活性，表明 Fe(II) 是 DOHH 的催化金属。铁与 DOHH 的化学计量以及铁结合和 DOHH 活性对四个保守 His-Glu 基序中每一个的依赖性表明，DOHH 单体具有一个双核含铁活性中心，就像其他非血红素铁依赖性单加氧酶，例​​如核糖核苷酸还原酶和甲烷单加氧酶。此外，添加2-酮戊二酸并不会增强人重组酶的DOHH活性，表明其反应机制不同于其他2-含氧酸和Fe(II)依赖性双加氧酶，例​​如脯氨酰4-羟化酶或赖氨酰羟化酶。 需要与编码 eIF5A、DHS 和 DOHH 的载体共转染，才能在哺乳动物细胞中过量产生含有马尿苷的活性 eIF5A。 为了确定克隆的人 DOHH 基因在培养的人细胞中是否有功能，我们使用哺乳动物表达载体表达人 DOHH ORF 与人 eIF5A-1 和 DHS ORF 组合。单独用p3XFLAG-CMV-7.1/heIF5A-1或与pCEFL/hDHS和pCEFL/hDOHH组合转染293T细胞。使用 eIF5A-1 抗体进行的蛋白质印迹显示，在所有用 p3XFLAG-CMV-7.1/heIF5A-1 转染的样品中，FLAG-eIF5A-1 过度表达。然而，在仅用FLAG-eIF5A-1载体转染的细胞中未检测到FLAG-eIF5A-1的标记，表明在这种情况下，FLAG-eIF5A-1蛋白主要作为未修饰的前体存在。仅在与修饰酶 DHS 共转染时，FLAG-eIF5A-1 的标记增强才显现出来。在这种情况下，Flag-eIF5A-1 的标记成分是脱氧马尿苷，而不是马尿苷，表明 DOHH 具有限制性。仅在 FLAG-eIF5A-1 载体与 DHS 和 DOHH 载体共转染后，才观察到 Flag-eIF5A-1 标记为 hypusine 形式。这些数据表明，克隆的人DOHH基因在哺乳动物细胞中表达功能性DOHH活性，并且所有三种蛋白质eIF5A、DHS和DOHH的共表达是在哺乳动物细胞中过量产生完全修饰的eIF5A所必需的。 eIF5A-1和eIF5A-2在人癌细胞中的差异表达 在报道的两种人类 eIF5A 基因中，通常只有一种异构体 (eIF5A-1) 在大多数人类细胞系中表达。 eIF5A-1 和 eIF5A-2 基因在哺乳动物细胞中差异表达。 eIF5A-1 mRNA 和蛋白质在所有人类细胞中组成型表达，而 eIF5A-2 mRNA (0.6-5.5 kb) 以组织特异性方式表达。仅在结直肠癌和卵巢癌来源的细胞系 SW-480 和 UACC-1598 中检测到 eIF5A-2 蛋白，这些细胞系显示 eIF5A-2 mRNA 的高过表达。具有不同长度的 3'-UTR 的多种形式的 eIF5A-2 mRNA 被鉴定为由于在 3'-UTR 中使用不同的聚腺苷酸化信号 (AATAAA) 而产生的一种基因的产物。