Universal pathway for posttransfer editing reactions: Insights from the crystal structure of TtPheRS with puromycin

At the amino acid binding and recognition step, phenylalanyl-tRNA synthetase (PheRS) faces the challenge of discrimination between cognate phenylalanine and closely similar noncognate tyrosine. Resampling of Tyr-tRNA^Phe to PheRS increasing the number of correctly charged tRNA molecules has recently been revealed. Thus, the very same editing site of PheRS promotes hydrolysis of misacylated tRNA species, associated both with cis- and trans-editing pathways. Here we report the crystal structure of Thermus thermophilus PheRS (TtPheRS) at 2.6 Å resolution, in complex with phenylalanine and antibiotic puromycin mimicking the A76 of tRNA acylated with tyrosine. Starting from the complex structure and using a hybrid quantum mechanics/molecular mechanics approach, we investigate the pathways of editing reaction catalyzed by TtPheRS. We show that both 2′ and 3′ isomeric esters undergo mutual transformation via the cyclic intermediate orthoester, and the editing site can readily accommodate a model of Tyr-tRNA^Phe where deacylation occurs from either the 2′- or 3′-OH. The suggested pathway of the hydrolytic reaction at the editing site of PheRS is of sufficient generality to warrant comparison with other class I and class II aminoacyl-tRNA synthetases.A key role in genetic code translation play aminoacyl-tRNA synthetases (aaRSs), providing linkage of amino acids to tRNAs. Before activation, at the amino acid recognition step, some aaRSs face a challenge of discrimination among amino acids with closely similar chemical structure. The rate of erroneous aminoacylation products generated in vivo is no more than one error per 10⁴−10⁵ correct reactions (1). To ensure such an extent of accuracy, aaRSs developed a multisieve mechanism of proofreading (2, 3). The existence of a proofreading activity has been demonstrated for both class I and class II aaRSs. Among aaRSs on record, about half of them are capable of selecting between amino acids resembling each other (4).The class aminoacyl-tRNA synthetases (aaRSs), namely IleRS, ValRS, and LeuRS, are characterized by a conserved connective polypeptide 1 (CP1) editing domain forming insertion into the catalytic core, except in cases of bacterial and mitochondrial LeuRSs, where the CP1 occurs at a different point of insertion (5). MetRS also falls into class I, but its CP1 lacks editing activity (6). The editing domains of class II aaRSs (ThrRS, ProRS, AlaRS, PheRS) are more diverse in amino acid sequence and in the distinguishing features of their folds. Kinetic experiments carried out for SerRS revealed the presence of a tRNA-independent pretransfer editing pathway (7).Detailed analyses of posttransfer editing were performed for class I LeuRS and class II ThrRS (8, 9). The structures of the LeuRS posttransfer complex imply the existence of water molecules that are specifically coordinated, to play the role of attacking nucleophiles. The alanine-scanning mutagenesis of the editing site has failed to identify key residues directly involved in catalysis (8). Thus, it was proposed that the CP1 domain simply binds the substrates in a configuration that favors attack by a water molecule, which itself is appropriately positioned by the set of key residues. The crystal structure of the editing domain from ThrRS complexed with Ser-A76 reveals two water molecules located on either side of the hydrolyzed bond (9). This study underlines the crucial role played by tRNA in substrate-assisted catalysis, in positioning the catalytic water molecules along with the protein side chains (9, 10).The 3D structures of Thermus thermophilus phenylalanyl-tRNA synthetase (TtPheRS) and its complexes with functional substrates (11–14) revealed that the catalytic α subunit exerts control over aminoacylation reaction whereas the major role of the β subunit lies in the recognition and binding of cognate tRNA^Phe and hydrolysis of misacylated tRNA (Fig. 1A). The early fast kinetic study demonstrated that tyrosine is indeed transferred to tRNA^Phe, and the misacylated tRNA is rapidly hydrolyzed (15). Later, it was established that editing activity of the bacterial and archaeal/eukaryotic PheRSs is associated with the active site located at the interface region between B3 and B4 domains in the β subunit (16–18).Open in a separate windowFig. 1.(A) Structure of the TtPheRS complex with puromycin and phenylalanine. The protein is shown in cartoon representation; the ligands puromycin (red) and phenylalanine (dark blue) are shown in space-filling representation. The domain architecture of one αβ-heterodimer is shown with the N-terminal coiled coil of the α-subunit colored cyan, catalytic domains A1 and A2 colored red, and structural domains of the β-subunit domains from B1 to B8 colored differently. The symmetry-related heterodimer is denoted with asterisks. (B) The editing site cavity of TtPheRS with bound puromycin. The electron density map (colored in red), calculated as described in SI Materials and Methods with coefficients (F_obs − F_calc) contoured at 2.5σ. Crystal structure of the TtPheRS complex in space-filling representation (colored gray) rendered to show protein surface interacting with puromycin.Here we present the crystal structure of TtPheRS, in complex with phenylalanine at the “synthetic” (aminoacylation) site and puromycin (mimicking the A76 of tRNA misacylated with Tyr) at the editing site. The natural substrate’s ester moiety represents an isoelectronic analog of the puromycin amide group, wherein the NH group is replaced with an ester oxygen atom. The appearance of puromycin at the editing site is accompanied by changes in the positions of some bound water molecules or even by their loss, compared with TtPheRS complex with Tyr (17). Loss of the water molecules, supposedly underlying the nucleophilic attack on the carbonyl carbon of the ester bond, gives grounds for revisiting the hydrolytic mechanism at work in bacterial PheRSs (17, 19). The suggested pathway of hydrolytic reaction is of sufficient generality to warrant comparison with those of other class I and class II aaRSs.     

Characterization of Fibroblast Growth Factor-2 Binding to Ribosomes

Abstract

We have examined the binding of FGF-2 to ribosomes and have found that in NIH 3T3 cells that synthesize high amounts of all FGF-2 forms, both 18 kDa and HMW forms of FGF-2 bind to ribosomes. Ribosomes purified from these cells were treated with RNase or puromycin to identify the binding site of FGF-2 on the ribosome. Neither RNase nor puromycin treatment affected the in vivo binding of FGF-2 to ribosomes suggesting that FGF-2 binds ribosomal protein or rRNA, but not mRNA. The stoichiometry of binding in these cells was approximately 1 FGF-2 molecule bound per 1 ribosome. Binding was unaffected by high salt treatment indicating that FGF-2 tightly associates with polysomes. An in vitro binding experiment performed with purified ribosomes and recombinant FGF-2 suggested that the binding site is saturable. HBNF, a protein with similar charge and size to FGF-2, bound 15-fold less than FGF-2 to purified ribosomes. These results indicate that the binding of FGF-2 to ribosomes is specific.     

Directed differentiation of regulatory T cells from naive T cells and prevention of their inflammation-mediated instability using small molecules

Regulatory T (T_reg) cell therapy is a promising approach for immune tolerance induction in autoimmunity conditions and cell/organ transplantations. Insufficient isolation yields and impurity during downstream processes and T_reg instability after adoptive transfer in inflammatory conditions are major limitations to T_reg therapy, and indicate the importance of seeking a valid, reliable method for de-novo generation of T_regs. In this research, we evaluated T_reg-like cells obtained from different T_reg differentiation protocols in terms of their yield, purity and activity. Differentiation was performed on naive CD4⁺ cells and a naive CD4⁺/T_reg co-culture by using three different protocols – ectopic expression of forkhead box protein P3 (E-FoxP3), soluble transforming growth factor β (S-TGF) and small molecules [N-acetyl puromycin and SR1555 (N-Ac/SR)]. The results showed that a high yield of a homogeneous population of T_reg-like cells could be achieved by the N-Ac/SR method under a T helper type 17 (Th17)-polarizing condition, particularly interleukin (IL)-6 and TGF-β, when compared with the E-FoxP3 and S-TGF methods. Surprisingly, SR completely inhibited the differentiation of IL-17-producing cells and facilitated T_reg generation in the inflammatory condition and had highly suppressive activity against T cell proliferation without T_reg-specific demethylase region (TSDR) demethylation. For the first time, to our knowledge, we report the generation of efficient, pure T_reg-like cells by using small molecules during in-vitro inflammatory conditions. Our results suggested that the N-Ac/SR method has several advantages for T_reg generation when compared with the other methods, including a higher purity of T_regs, easier procedure, superior suppressive activity during the inflammatory condition and decreased cost.     

Anti-apoptotic effect of HIV protease inhibitors via direct inhibition of calpain

Treatment with drugs designed to inhibit the HIV protease ameliorates immune functions in AIDS patients, reducing cell deletion by apoptosis even in the absence of inhibition of viral spread. This suggests that they interact with the intrinsic apoptotic signaling. We found that caspases, the main executioner of the apoptotic process, are not directly inhibited. In search for the mechanism responsible for their anti-apoptotic effect, we have found that indinavir and ritonavir are able to inhibit apoptosis only in those cell systems where apoptosis involves the activation of calpains. They directly inhibit a calpain-like activity expressed in lysates from apoptotic cells, to the same extent as commercially available calpain inhibitor 1. In in vitro assays with purified calpains, indinavir and ritonavir strongly inhibit m-calpain, and moderately mu-calpain. These results have great therapeutic implications, going beyond AIDS treatment, since many degenerative disorders involve abnormal calpain activation, indicating calpain as an ideal pharmacological target. Indinavir and ritonavir, potent m-calpain inhibitors, largely used since several years on humans without important negative side effects, may become powerful tools against those pathologies.     

肾炎灵颗粒剂治疗慢性原发性肾小球疾病研究(1)——临床部分

运用肾炎灵颗粒剂治疗慢性原发性肾小球疾病 195例 ,其中 5 6例肾穿刺明确病理诊断 ,并设阳性中药对照组。治疗结果表明 ,肾炎灵颗粒剂能明显降低患者的蛋白尿、红细胞尿 ,并能改善肾小管间质和肾功能损害。治疗各类慢性原发性肾小球疾病的总有效率为 87 6% ,与阳性中药对照组 5 9 3 8%的总有效率对比差异有显著意义 (P <0 0 5 ) ,同时能明显改善本病的主要症状 ,未发现有影响治疗的明显副反应。实验 ( 1)结果提示 ,肾炎灵颗粒剂可明显降低嘌呤霉素肾病大鼠的尿蛋白定量 ,降低血胆固醇及甘油三酯含量 ,减轻肾脏组织病理损害。实验 ( 2 )结果提示 ,喂服肾炎灵颗粒剂大鼠的血清 ,对培养的系膜细胞3H thymidine的掺入率的抑制作用 ,与小牛血清培养液对比 (P <0 0 1) ,而且在 0 5 %低浓度时 ,即显示有明显的抑制作用。对成纤维细胞的3H thymidine掺入率的抑制作用 ,在 10 %、2 0 %浓度时 ,与 10 %、2 0 %未喂药大鼠血清培养液对照组对比 (P <0 0 1)。     

单次静脉注射嘌呤霉素氨基核苷所致大鼠肾病模型的优化及评价

目的明确大鼠嘌呤霉素氨基核苷(puromycinaminonucleoside,PAN)肾病模型的适宜条件和病程演变,为该模型更好地用于肾病综合征、肾硬化发病机制及治疗的研究奠定基础。方法用6周龄雄性Wistar大鼠,一次性颈静脉注射PAN,分别以2、3、4、5、7、9mg/100g体重的剂量给药,以等量生理盐水作对照,测定2周内不同时间点的尿蛋白排泄量;另以9mg/100g体重的剂量给药,观察28周内尿蛋白、血清胆固醇和三酰甘油的变化及肾组织的病理改变。结果各个剂量的PAN均可引起不同程度的蛋白尿,约在10~14d达到高峰,最高达(592.0±61.0)mg/24h。28周连续观察的结果显示,PAN组动物的尿蛋白呈现典型的双相曲线,即第2周达到高峰,伴有血清胆固醇及三酰甘油的升高,此后逐渐降至接近正常,但自第12周起尿蛋白再度逐渐上升,第28周可达急性期峰值的一半。结论6周龄雄性Wistar大鼠,一次性颈静脉注射适量PAN可建立不同程度的肾病模型,成功率极高,方法简单,用药量省,动物价廉易得,且急性期发病快而周期短,不失为研究人类相应疾病的良好模型。     

Lipoproteins in experimental nephrosis: plasma levels and composition.

Experimental nephrosis was induced in rats by administration of puromycin aminonucleoside and the levels of plasma lipoproteins were examined 7 days later and compared to controls. As determined by density ultracentrifugation, VLDL, IDL, LDL, and HDL protein levels were increased by 8, 4, 5, and 5 times, respectively. These increases were accompanied by changes in lipid and apoprotein composition. The VLDL, IDL, and LDL fractions contained less triglyceride and more phospholipid and cholesterol, while HDL lipid composition was not altered. The apoprotein composition of VLDL and IDL were not measurably altered, but LDL contained less apoE. HDL had a markedly abnormal composition characterized by an almost complete absence of apoA-IV and apoE, increased apoA-1, and decreased apoC. While increased hepatic synthesis can account for much of the observed hyperlipoproteinemia in nephrosis, the changes in lipoprotein composition suggest impaired catabolism as a contributory factor.     

CD2AP在大鼠肾病模型中的表达及意义

目的：观察CD2-associated protein(CD2AP)在大鼠氨基核苷肾病模型。肾小球中的表达变化及其意义。方法：通过建立大鼠氨基核苷肾病模型，采用免疫组织化学技术观察CD2AP在不同时间点。肾病模型大鼠。肾小球中的表达和分布变化。结果：①。肾小球足细胞CD2AP的表达在。肾病模型建立的早期即有下调；在大鼠肾病模型蛋白尿的高峰期，CD2AP的表达明显下降；在肾病模型大鼠的疾病恢复期，CD2AP的表达逐步恢复正常；②肾小球足细胞CD2AP表达量的变化与肾病模型大鼠24h尿蛋白定量的变化存在着负相关。结论：①CD2AP是判断。肾小球足细胞损伤的一个早期重要指标；②CD2AP在足细胞中表达量的改变可能是肾小球滤过屏障功能异常的病理生理基础。