Combined tRNA modification defects impair protein homeostasis and synthesis of the yeast prion protein Rnq1

Modified nucleosides in tRNA anticodon loops such as 5-methoxy-carbonyl-methyl-2-thiouridine (mcm⁵s²U) and pseuduridine (Ψ) are thought to be required for an efficient decoding process. In Saccharomyces cerevisiae, the simultaneous presence of mcm⁵s²U and Ψ38 in tRNA^Gln_UUG was shown to mediate efficient synthesis of the Q/N rich [PIN⁺] prion forming protein Rnq1.¹ Klassen R, Ciftci A, Johanna Funk J, Bruch A, Butter F, Schaffrath R. tRNA anticodon loop modifications ensure protein homeostasis and cell morphogenesis in yeast. Nucleic Acids Res 2016; 44(22):10946-959. pii: gkw705; PMID:27496282; http://dx.doi.org/10.1093/nar/gkw705[Crossref], [PubMed], [Web of Science ®] [Google Scholar] In the absence of these two tRNA modifications, higher than normal levels of hypomodified tRNA^Gln_UUG, but not its isoacceptor tRNA^Gln_CUG can restore Rnq1 synthesis. Moroever, tRNA overexpression rescues pleiotropic phenotypes that associate with loss of mcm⁵s²U and Ψ38 formation. Notably, combined absence of different tRNA modifications are shown to induce the formation of protein aggregates which likely mediate severe cytological abnormalities, including cytokinesis and nuclear segregation defects. In support of this, overexpression of the aggregating polyQ protein Htt103Q, but not its non-aggregating variant Htt25Q phenocopies these cytological abnormalities, most pronouncedly in deg1 single mutants lacking Ψ38 alone. It is concluded that slow decoding of particular codons induces defects in protein homeostasis that interfere with key steps in cytokinesis and nuclear segregation.     

Interaction of aminoacyl-tRNA synthetases with tRNA: General principles and distinguishing characteristics of the high-molecular-weight substrate recognition

This review summarizes results of numerous (mainly functional) studies that have been accumulated over recent years on the problem of tRNA recognition by aminoacyl-tRNA synthetases. Development and employment of approaches that use synthetic mutant and chimeric tRNAs have demonstrated general principles underlying highly specific interaction in different systems. The specificity of interaction is determined by a certain number of nucleotides and structural elements of tRNA (constituting the set of recognition elements or specificity determinants), which are characteristic of each pair. Crystallographic structures available for many systems provide the details of the molecular basis of selective interaction. Diversity and identity of biochemical functions of the recognition elements make substantial contribution to the specificity of such interactions.     

Whole Genome Phage Display Selects for Proline-rich Boi Polypeptides against Bem1p

Interaction selection by biopanning from a fragmented yeast proteome displayed on filamentous phage particles was successful in identifying proline-rich fragments of Boi1p and Boi2p. These proteins bind to the second ``src homology region 3' (SH3) domain of Bem1p, a protein of Saccharomyces cerevisiae involved in bud formation. Target Bem1p was a doubly-tagged recombinant, Bem1_{[Asn142-Ile551]}, which strongly interacts in ELISA with a C-terminal 75 amino acids polypeptide from Cdc24p exposed on phage. The whole yeast genomic display library contained ~7.7 × 10⁷ independent clones of sheared S. cerevisiae genomic DNA fused to a truncated M13 gene III. This study corroborates the value of fragmented-proteome display to identify strong and direct interacting protein modules.     

Phage Display and Synthetic Peptides as Promising Biotechnological Tools for the Serological Diagnosis of Leprosy

Background

The diagnosis of leprosy is primarily based on clinical manifestations, and there is no widely available laboratory test for the early detection of this disease, which is caused by Mycobacterium leprae. In fact, early detection and treatment are the key elements to the successful control of leprosy.

Methodology/Principal Findings

Peptide ligands for antibodies from leprosy patients were selected from phage-displayed peptide libraries. Three peptide sequences expressed by reactive phage clones were chemically synthesized. Serological assays that used synthetic peptides were evaluated using serum samples from leprosy patients, household contacts (HC) of leprosy patients, tuberculosis patients and endemic controls (EC). A pool of three peptides identified 73.9% (17/23) of multibacillary (MB) leprosy patients using an enzyme-linked immunosorbent assay (ELISA). These peptides also showed some seroreactivities to the HC and EC individuals. The peptides were not reactive to rabbit polyclonal antisera against the different environmental mycobacteria. The same peptides that were conjugated to the carrier protein bovine serum albumin (BSA) induced the production of antibodies in the mice. The anti-peptide antibodies that were used in the Western blotting analysis of M. leprae crude extracts revealed a single band of approximately 30 kDa in one-dimensional electrophoresis and four 30 kDa isoforms in the two-dimensional gel. The Western blotting data indicated that the three peptides are derived from the same bacterial protein.

Conclusions/Significance

These new antigens may be useful in the diagnosis of MB leprosy patients. Their potentials as diagnostic reagents must be more extensively evaluated in future studies using a large panel of positive and negative sera. Furthermore, other test approaches using peptides should be assessed to increase their sensitivity and specificity in detecting leprosy patients. We have revealed evidence in support of phage-displayed peptides as promising biotechnological tools for the design of leprosy diagnostic serological assays.     

A Protocol for Phage Display and Affinity Selection Using Recombinant Protein Baits

Using recombinant phage as a scaffold to present various protein portions encoded by a directionally cloned cDNA library to immobilized bait molecules is an efficient means to discover interactions. The technique has largely been used to discover protein-protein interactions but the bait molecule to be challenged need not be restricted to proteins. The protocol presented here has been optimized to allow a modest number of baits to be screened in replicates to maximize the identification of independent clones presenting the same protein. This permits greater confidence that interacting proteins identified are legitimate interactors of the bait molecule. Monitoring the phage titer after each affinity selection round provides information on how the affinity selection is progressing as well as on the efficacy of negative controls. One means of titering the phage, and how and what to prepare in advance to allow this process to progress as efficiently as possible, is presented. Attributes of amplicons retrieved following isolation of independent plaque are highlighted that can be used to ascertain how well the affinity selection has progressed. Trouble shooting techniques to minimize false positives or to bypass persistently recovered phage are explained. Means of reducing viral contamination flare up are discussed.     

噬菌体短肽库的构建及其随机短肽的多样性

噬菌体短肽库是将随机合成的寡核苷酸序列通过与单链噬菌体外壳蛋白基因融合,从而将随机短肽表达于噬菌体的表面。将体外随机化学合成的寡聚核苷酸序列重组到单价噬菌体表达载体,构建了噬菌体短肽库,证明其库容为２×１０￣７集落形成单位（ｃｆｕ）,重组率为９３％。同时将１１个随机克隆进行序列测定,证实其寡聚核苷酸序列和氨基酸的分布几乎是完全随机的,其多样性可以满足特异性短肽筛选的要求。     

Direct Binding between Pre-S1 and TRP-like Domains in TRPP Channels Mediates Gating and Functional Regulation by PIP2

1. Download high-res image (152KB)
2. Download full-size image

     

噬菌体展示 protein A 及 protein L Ig 结合单结构域随机组合文库及 Ig 亲和筛选

Protein A 和 protein L 是细菌产生的两种结构和功能均不同的免疫球蛋白 (immunoglobulin , Ig) 结合分子,在细菌的致病中起重要作用 . 用含 SacⅠ位点的特定引物 PCR 分别扩增制备 protein A 的 A 、 B 、 C 、 D 抗体结合结构域和 protein L 的 B3 抗体结合结构域,各结构域 DNA 片段经 SacⅠ酶切后,再随机连接形成各种不同长度的分子组合文库,将该文库呈现在噬菌体表面构建了噬菌体展示 Ig 结合分子单结构域随机组合文库,所建组合文库容量为 2.3×10⁶个菌落形成单位,滴度为 4.1×10¹¹TU/ml , 包含各种单结构域片段,并以随机方式连接 . 用人 Ig 对该文库进行 4 轮亲和筛选,随机挑选 36 个代表性的阳性克隆进行序列测定分析表明,亲和筛选获得了多种非天然形式存在的新的 Ig 结合分子结构,其中 32 个克隆具有由 protein L 的单结构域和 protein A 的单结构域间隔重复排列而成的特征性 (MDPL-MDPA)n 结构 . 对噬菌体展示 Ig 结合分子单结构域随机组合文库的体外分子进化研究的尝试,为 Ig 结合分子的结构和功能研究提供了一新的途径,也为 Ig 结合分子的定向改造打下基础 .     

双峰驼源天然噬菌体纳米抗体展示库的构建及抗GDH纳米抗体筛选

目的:构建噬菌体天然纳米抗体展示库,以期用于筛选不同抗原分子的纳米抗体筛选平台,并用艰难梭菌谷氨酸脱氢酶(GDH)抗原筛选靶向GDH的纳米抗体,对所构建的噬菌体天然纳米抗体展示库进行验证。方法:采用Oligo DT提取双峰骆驼脾脏总RNA进行反转录,通过巢氏PCR获取全套重链可变区基因,将其构建到噬菌粒pCANTAB5E载体,经多次电转化至E. coil TG1构建初级噬菌体抗体库,经辅助噬菌体拯救后构成噬菌体展示库,并对噬菌体展示库的库容及多样性进行分析和鉴定。同时以GDH为靶向抗原对文库进行淘筛,计算淘筛回收率,并对第三轮淘筛后平板的单克隆进行ELISA鉴定。结果:构建的天然噬菌体纳米抗体库的插入率为95%左右,随机挑取的9个克隆氨基酸同源性为66. 17%,经MEGA分析后具有较好的多样性,同时经辅助噬菌体拯救后,得到的噬菌体展示库滴度为4×10~(12)CFU/ml。在三轮淘筛过程中,回收率逐步升高,噬菌体得到了有效的富集,同时对阳性克隆进行测序及分析,最终得到2条抗GDH纳米抗体序列。结论:成功构建了双峰驼源天然噬菌体纳米抗体展示文库且多样性良好,为后续筛选其他的靶向抗原奠定了基础,同时筛选获得两条抗GDH纳米抗体序列,为制备艰难梭菌谷氨酸脱氢酶诊断抗体提供技术支撑。     

噬菌体呈示单链抗体表达载体及小鼠非特异抗体库的构建

用大肠杆菌丝状噬菌体表面呈示技术构建抗体库的方法,为从抗原出发获得特异抗体提供了新的途径。报道的是,首先构建了一个用于噬菌体呈示抗体的噬粒表达载体ｐＦＵＷ８０,它具有既可以进行外分明表达,又可进行附着表达的特点。然后利用设计的一套扩增小鼠抗体重链和轻链可变区基因片段的ＰＣＲ引物,从未免疫小鼠脾细胞中扩增出了抗体重、轻链可变区基因,构建了一个１．２×１０６库容的小鼠非特异性单链抗体库。从这个抗体库中,筛选出了针对人ＩｇＧ的单链抗体噬菌体,并进行了ＥＬＩＳＡ检测和部分序列分析。这一初步结果为今后继续利用这一系统进行研究奠定了基础。     

Effect of the sulphur atom on geometry and spectra of the biomolecule 2-thiouracil and in the WC base pair 2-thiouridine-adenosine. Influence of water in the first hydration shell

The effect of the sulphur atom on 2-thiouracil (2TU) and 2-thiouridine molecules, as compared with uracil and uridine molecules, respectively, was carried out in several environments. The predicted IR spectrum of 2TU in the isolated state was compared with that obtained for uracil molecule and with those reported experimentally in matrix isolation. Its crystal unit cell in the solid state was simulated through a tetramer form using DFT methods for the first time. The calculated Raman spectrum was compared to the experimental ones in the solid state. A linear scaling procedure was used for this task. The first hydration shell was simulated by explicit number of water molecules surrounding 2TU up to 30 and was compared with that obtained in uracil molecule. Water molecules ‘distributed’ around 2TU was preferred over that ‘clustering’, because it can better reproduce the hydration and their effects on different parameters of the molecular structure of 2TU and uracil. The total atomic charges and several calculated thermodynamic parameters were discussed. The effect of the sulphur atom on the Watson-Crick (WC) and reverse WC base pair uridine-adenosine was estimated, and the CP corrected interaction energies were calculated. 2-thiouridine has a weaker WC pair than that with uridine, although its slight higher dipole moment (μ) facilitates the interaction with the water molecules. Several helical parameters were determined.     

Domains of the human androgen receptor and glucocorticoid receptor involved in binding to the nuclear matrix

Steroid receptors have been reported to bind to the nuclear matrix. The nuclear matrix is operationally defined as the residual nuclear structure that remains after extraction of most of the chromatin and all soluble and loosely bound componnets. To obtain insight in the molecular mechanism of the interaction of steroid receptors with the nuclear matrix, we studied the binding of several deletion mutants of the human androgen receptor (hAR) and the human glucocorticoid receptor (hGR) to the nuclear matrix. Receptor binding was tested for two different nuclear matrix preparations: complete matrices, in which most matrix proteins are retained during the isolation procedure, and depleted matrices, which consist of only a subset of these proteins. The results show that the C-terminal domain of the hAR binds tightly to both depleted and complete matrices. In addition, at least one other domain of the hAR binds to complete matrices but not to depleted matrices. In contrast to the hAR, the hGR binds only to complete matrices. For this interaction both the DNA-binding domain and the C-terminal domain of the hGR are required, whereas the N-terminal domain is not. We conclude that specific protein domains of the hAR and the hGR are involved in binding to the nuclear matrix. In addition, our results indicate that the hAR and the hGR are attached to the nuclear matrix through different molecular interactions.     

Codon:Anticodon and anticodon:Anticodon interaction. Evaluation of equilibrium and kinetic parameters of complexes involving a G:U wobble

In order to learn about the effect of the G:U wobble interaction we characterized the codon:anticodon binding between triplets: UUC, UUU and yeast tRNA^Phe (anticodon GmAA) as well as the anticodon:anticodon binding between Escherichia coli tRNA^Glu₂, E. coli tRNA^Lys (anticodons: mam⁵s²UUC, and mam⁵s²UUU, respectively) and tRNA^Phe from yeast and E. coli (anticodon GAA) using equilibrium fluorescence titrations and temperature jump measurements with fluorescence and absorption detection. The difference in stability constants between complexes involving a G:U pair rather than a usual G:C basepair is in the range of one order of magnitude and is mainly due to the shorter lifetime of the complex involving G:U in the wobble position. This difference is more pronounced when the codon triplet is structured, i.e., is built in the anticodon loop of a tRNA. The reaction enthalpies of the anticodon:anticodon complexes involving G:U mismatching were found to be about 4 kcal/mol smaller, and the melting temperatures more than 20°C lower, than those of the corresponding complexes with the G:C basepair. The results are discussed in terms of different strategies that might be used in the cell in order to minimize the effect of different lifetimes of codon-tRNA complexes. Differences in these lifetimes may be used for the modulation of the translation efficiency.     

7肽和12肽两种M13噬菌体展示库筛选肿瘤坏死因子alpha拮抗肽的比较

肿瘤坏死因子alpha的拮抗剂是治疗多种炎症性自身免疫疾病的首选,但抗体类拮抗物因副作用明显而使用受限,尤其是机体内抗抗体的产生,严重影响治疗效果和药物代谢。而肽类物除免疫原性低之外,和小分子相比也有更低的毒性和更强的靶标特异性。使用7肽和12肽两种M13噬菌体展示库筛选TNFα拮抗肽,以分析7肽和12肽分别作为TNFα拮抗肽的亲和性与功能性。经过3~4轮的筛选和验证,得到2条7肽序列和2条12肽序列。利用ELISA方法检测合成肽与TNFα结合的亲和性,编号632的7肽亲和性最强,Kd=138nmol/L;编号636的12肽亲和性稍差,Kd=8.59μmol/L。InsightⅡ软件分别分析632肽和636肽与TNFα二聚体结合,发现632肽与TNFα二聚体结合更加稳定,并且在细胞水平上632肽拮抗TNFα活性功能比636肽更强,有632肽存在的条件下TNFα诱导的L929细胞生存率上升了3倍,而636肽的作用只有2倍。7肽比12肽更适合作为TNFα拮抗肽。     

脂多糖保守表位模拟肽的筛选与鉴定

用针对脂多糖保守表位的单抗2B4对噬菌体随机12肽库进行亲和筛选,通过噬菌体ELISA实验及脂多糖(LPS)竞争抑制实验鉴定阳性克隆.经三轮筛选后,与抗体结合的噬菌体得到明显富集,噬菌体ELISA结果显示,阳性率达80%.将其中12个阳性噬菌体克隆做鼠伤寒杆菌和大肠杆菌LPS竞争抑制实验,抑制作用非常明显,有良好的剂量依赖关系,证明这12个克隆与LPS具相似表位.DNA测序并推导噬菌体展示肽的氨基酸序列为,GPPQWFFSQPQL（5/12,41.7%）,LPQYFWNTATTA （3/12,25%）,FPQNHWNVPWAT（2/12,16.6%）,HSQSFWNAPLAM和AHPWTHGYFPPL（1/12,8.3%）.实验结果表明,用2B4抗体筛选到的噬菌体短肽克隆可模拟保守表位,即脂多糖的模拟肽（位）.     

脂多糖保守表位模拟肽的筛选与鉴定

用针对脂多糖保守表位的单抗2B4对噬菌体随机12肽库进行亲和筛选,通过噬菌体ELISA实验及脂多糖(LPS)竞争抑制实验鉴定阳性克隆.经三轮筛选后,与抗体结合的噬菌体得到明显富集,噬菌体ELISA结果显示,阳性率达80%.将其中12个阳性噬菌体克隆做鼠伤寒杆菌和大肠杆菌LPS竞争抑制实验,抑制作用非常明显,有良好的剂量依赖关系,证明这12个克隆与LPS具相似表位.DNA测序并推导噬菌体展示肽的氨基酸序列为,GPPQWFFSQPQL（5/12,41.7%）,LPQYFWNTATTA（3/12,25%）,FPQNHWNVPWAT（2/12,16.6%）,HSQSFWNAPLAM和AHPWTHGYFPPL（1/12,8.3%）.实验结果表明,用2B4抗体筛选到的噬菌体短肽克隆可模拟保守表位,即脂多糖的模拟肽（位）.     

Regulation of Bone Morphogenetic Protein Activity by Pro Domains and Proprotein Convertases

Bone morphogenetic proteins (BMPs) are derived from inactive precursor proteins by endoproteolytic cleavage. Here we show that processing of Nodal and Myc-tagged BMP4 is significantly enhanced by SPC1/Furin or SPC4/PACE4, providing direct evidence that regulation of BMP signaling is likely to be controlled by subtilisin-like proprotein convertase (SPC) activities. Nodal processing is dramatically enhanced if two residues adjacent to the precursor cleavage site are substituted with amino acids found at the equivalent positions of Activin, demonstrating that structural constraints at the precursor cleavage site limit the processing efficiency. However, in transfection assays, mature Nodal is undetectable either in culture supernatants or in cell lysates, despite efficient cleavage of the precursor protein, suggesting that mature Nodal is highly unstable. Domain swap experiments support this conclusion since mature BMP4 or Dorsalin are also destabilized when expressed in conjunction with the Nodal pro domain. By contrast, mature Nodal is stabilized by the Dorsalin pro domain, which mediates the formation of stable complexes. Collectively, these data show that the half-life of mature BMPs is greatly influenced by the identity of their pro regions.