Duplication and Functional Specialization of the Telomere-capping Protein Cdc13 in Candida Species

The budding yeast G-tail binding complex CST (Cdc13-Stn1-Ten1) is crucial for both telomere protection and replication. Previous studies revealed a family of Cdc13 orthologues (Cdc13A) in Candida species that are unusually small but are nevertheless responsible for G-tail binding and the regulation of telomere lengths and structures. Here we report the identification and characterization of a second family of Cdc13-like proteins in the Candida clade, named Cdc13B. Phylogenetic analysis and sequence alignment indicate that Cdc13B probably arose through gene duplication prior to Candida speciation. Like Cdc13A, Cdc13B appears to be essential. Deleting one copy each of the CDC13A and CDC13B genes caused a synergistic effect on aberrant telomere elongation and t-circle accumulation, suggesting that the two paralogues mediate overlapping and nonredundant functions in telomere regulation. Interestingly, Cdc13B utilizes its C-terminal OB-fold domain (OB4) to mediate self-association and binding to Cdc13A. Moreover, the stability of the heterodimer is evidently greater than that of either homodimer. Both the Cdc13 A/A homodimer and A/B heterodimer, but not the B/B homodimer, recognized the telomere G-tail repeat with high affinity and sequence specificity. Our results reveal novel evolutionary elaborations of the G-tail-binding protein in Saccharomycotina yeast, suggesting a drastic remodeling of CDC13 that entails gene duplication, fusion, and functional specialization. The repeated and independent duplication of G-tail-binding proteins such as Cdc13 and Pot1 hints at the evolutionary advantage of having multiple G-tail-binding proteins.     

Increase of anti-HIV activity of C-peptide fusion inhibitors using a bivalent drug design approach

We reported the design of fusion inhibitors with improved activity using a multivalent inhibitor design strategy. First, we chose C29 as the template sequence, which is a 29-mer peptide derived from HIV-1 gp41 CHR domain and has anti-HIV activity of IC₅₀ 118 nM in a cell–cell fusion assay. We optimized the crosslink sites and linkers of the template peptide. We found that N-terminal crosslink caused activity improvement based on the multivalent co-operative effect. Especially, the IC₅₀ of peptide (CAcaC29)₂ was improved from 49.02 (monomeric form) to 5.71 nM. Compared with long peptides, short peptides may be more suitable to analyze the co-operative effect. So we selected a shorter peptide C22 to synthesize the bivalent inhibitors. Due its weak helicity, no co-operative effect appeared. Therefore, we chose SC22EK, which were introduced salt bridges to consolidate the helicity based on the natural sequence C22. The cross-linked (CAcaSC22EK)₂ was four times more potent than the monomer SC22EK in anti-HIV activity, with an IC₅₀ value of 4.92 nM close to the high active peptide fusion inhibitor C34. The strategy used in this study may be used to design new fusion inhibitors to interfere similar processes.     

Dimeric argininamide-type neuropeptide Y receptor antagonists: Chiral discrimination between Y1 and Y4 receptors

The structurally related peptides neuropeptide Y (NPY), peptide YY (PYY) and pancreatic polypeptide (PP) are endogenous agonists of the NPY receptor (YR) family, which in humans comprises four functionally expressed subtypes, designated Y₁R, Y₂R, Y₄R and Y₅R. Nonpeptide antagonists with high affinity and selectivity have been described for the Y₁R, Y₂R and Y₅R, but such compounds are still lacking for the Y₄R. In this work, the structures of the high affinity selective (R)-argininamide-type Y₁R antagonists BIBP3226 and BIBO3304 were linked via the guanidine or urea moieties to give homo-dimeric argininamides with linker lengths ranging from 31 to 41 atoms. Interestingly, the twin compounds proved to be by far less selective for the Y₁R than the R-configured monovalent parent compounds. The decrease in selectivity ratio was most pronounced for Y₁R versus Y₄R subtype, resulting in comparable affinities of bivalent ligands for Y₁R and Y₄R (e.g. UR-MK177 ((R,R)-49): K_i = 230 nM (Y₁R) and 290 nM (Y₄R)). With a K_i value of 130 nM and a K_b value of 20 nM, UR-MK188 ((R,R)-51) was superior to all Y₄R antagonists known to date. The S,S-configured optical antipodes of UR-MK177 and UR-MK188 (UR-MEK381 ((S,S)-49) and UR-MEK388 ((S,S)-51)) were synthesized to investigate the stereochemical discrimination by the different receptor subtypes. Whereas preference for R,R-configured argininamides was characteristic of the Y₁R, stereochemical discrimination by the Y₄R was not observed. This may pave the way to selective Y₄R antagonists.     

Novel functional variants locus in PLCE1 and susceptibility to esophageal squamous cell carcinoma: Based on published genome-wide association studies in a central Chinese population

A novel functional single nucleotide polymorphism (SNP) rs2274223 located in the phospholipase C epsilon 1 (PLCE1) gene was found to be associated with the risk of esophageal squamous cell carcinoma (ESCC) by three large-scale genome-wide association studies (GWAS) in Chinese populations. In the present study, we validated this finding and also explored the risk of ESCC associated with other two unreported potentially functional SNPs (rs17417407 G > T and rs2274224 C > G) of PLCE1 in a population-based case–control study to investigate the association between these three potentially functional SNPs in PLCE1 and susceptibility to ESCC. A total of 381 ESCC cases and 420 controls matched by age and sex were recruited and successfully genotyped for three SNPs (rs17417407, rs2274223 and rs2274224) of the PLCE1 in a central Chinese population. SNP rs2274223 was independently associated with increased risk of ESCC (adjusted odds ratio [OR], 2.80; 95% confidence interval [95% CI], 1.45–5.39 for GG vs. AA), and SNP rs2274224 was found to be associated with decreased risk of ESCC (adjusted OR, 0.65; 95% CI, 0.46–0.91 for CG vs. CC). The combined effects of risk alleles for three SNPs (rs17417407T, rs2274223G and rs2274224G) were found to be associated with elevated risk of ESCC in a dose-dependent effect manner (P_trend = 0.005). The G_rs17417407A_rs2274223C_rs2274224 haplotype decreased the risk of ESCC (adjusted OR, 0.76; 95% CI, 0.62–0.93), meanwhile the G_rs17417407G_rs2274223C_rs2274224 and T_rs17417407G_rs2274223C_rs2274224 haplotypes could increase the risk of ESCC (adjusted OR, 1.75; 95% CI, 1.33–2.18 and OR, 2.51; 95% CI, 1.15–2.49). Gene–environment interaction analysis presented a best model consisted of four factors (rs2274223, rs2274224, family history, and smoking) with testing balance accuracy (TBA): 0.66 and cross validation consistency (CVC): 7/10, which could increase the esophageal cancer risk in the “high risk group” with 3.67-fold (OR: 3.67, 95% CI: 2.74–4.92), compared to the “low risk group”. Our results further confirmed that genetic variations in PLCE1 may contribute to ESCC risk associated with tobacco exposure in a central Chinese population. Further functional studies are needed to validate our results.     

基于SNP 互作识别潜在冠心病致病基因

目的:基于全基因组关联分析(Genomewideassociationstudy,GWAS)数据与生物信息学方法,识别冠心病潜在致病基因。方法:利用生物信息学方法和GWAS数据,对单核苷酸多态性(SingleNucleotidePolymorphisms,SNP)进行疾病风险打分,依据特定距离阈值内的SNP-SNP互作关系,筛选出疾病相关SNP显著风险模块,识别潜在致病基因。结果:设定阈值20kb,经筛选获得279个SNP显著风险模块,映射到79个基因,文献验证率为71.01%。结论:基于SNP互作识别的潜在致病基因,能更加准确的分析冠心病的发生发展过程。     

A Bipartite Network-based Method for Prediction of Long Non-coding RNA–protein Interactions

As one large class of non-coding RNAs(nc RNAs), long nc RNAs(lncRNAs) have gained considerable attention in recent years. Mutations and dysfunction of lnc RNAs have been implicated in human disorders. Many lnc RNAs exert their effects through interactions with the corresponding RNA-binding proteins. Several computational approaches have been developed, but only few are able to perform the prediction of these interactions from a network-based point of view. Here,we introduce a computational method named lnc RNA–protein bipartite network inference(LPBNI). LPBNI aims to identify potential lnc RNA–interacting proteins, by making full use of the known lnc RNA–protein interactions. Leave-one-out cross validation(LOOCV) test shows that LPBNI significantly outperforms other network-based methods, including random walk(RWR)and protein-based collaborative filtering(Pro CF). Furthermore, a case study was performed to demonstrate the performance of LPBNI using real data in predicting potential lnc RNA–interacting proteins.     

利用酵母双杂交系统筛选与黄瓜花叶病毒外壳蛋白互作的寄主蛋白

利用酵母双杂交系统,以黄瓜花叶病毒(Cucumber mosaic virus,CMV)的外壳蛋白(coat protein,CP)为诱饵,从番茄叶片c DNA文库中筛选与其互作的蛋白。结果显示,诱饵载体pBT3-SUC-CMV-CP均能在酵母细胞中正确表达,无自激活活性而且对酵母无毒性;通过对酵母双杂交文库的筛选和回转验证,共获得了98个阳性克隆,分别编码67个可能与CMV-CP相互作用的蛋白,分别参与植物防御反应、光合作用、物质转运、信号转导、能量代谢、氨基酸代谢、细胞壁的形态建成、植物的激素代谢等。本研究结果表明,CMV CP可同时调控寄主的多个代谢过程,在CMV的致病过程中有多重功能。     

Interaction of cobalt(II) and copper(II) hydroxamates with polyriboadenylic acid: An insight into RNA based drug designing

The polyadenylic acid [poly(A)] tail of mRNA plays a noteworthy role in the initiation of the translation, maturation, and stability of mRNA. It also significantly contributes to the production of alternate proteins in eukaryotic cells. Hence, it has recently been recognized as a prospective drug target. Binding affinity of bis(N-p-tolylbenzohydroxamato)Cobalt(II), [N-p-TBHA-Co(II)] (1) and bis(N-p-naphthylbenzohydroxamato)Copper(II), [N-p-NBHA-Cu(II)] (2) complexes with poly(A) have been investigated by biophysical techniques namely, absorption spectroscopy, fluorescence spectroscopy, diffuse reflectance infrared Fourier transform spectroscopy, circular dichroism spectroscopy, viscometric measurements and through molecular docking studies. The intrinsic binding constants (K_b) of complexes were determined following the order of N-p-TBHA-Co(II)] > N-p-NBHA-Cu(II), along with hyperchromism and a bathochromic shift for both complexes. The fluorescence quenching method revealed an interaction between poly(A)-N-p-TBHA-Co(II)/poly(A)-N-p-NBHA-Cu(II). The mode of binding was also determined via the fluorescence ferrocyanide quenching method. The increase in the viscosity of poly(A) that occurred from increasing the concentration of the N-p-TBHA-Co(II)/N-p-NBHA-Cu(II) complex was scrutinized. The characteristics of the interaction site of poly(A) with N-p-TBHA-Co(II)/N-p-NBHA-Cu(II) were adenine and phosphate groups, as revealed by DRS-FTIR spectroscopy. Based on these observations, a partial intercalative mode of the binding of poly(A) has been proposed for both complexes. Circular dichroism confirmed the interaction of both the complexes with poly(A). The molecular docking results illustrated that complexes strongly interact with poly(A) via the relative binding energies of the docked structure as ?259.39eV and ?226.30eV for N-p-TBHA-Co(II) and N-p-NBHA-Cu(II) respectively. Moreover, the binding affinity of N-p-TBHA-Co(II) is higher in all aspects than N-p-NBHA-Cu(II) for poly(A).     

1H NMR investigation on interaction between ibuprofen and lipoproteins

A large number of studies indicate that oxidative modification of plasma lipoproteins, especially low-density lipoprotein (LDL), is a critical factor in initiation and progression of atherosclerosis. We have previously found that ibuprofen (IBP), a potential antioxidant drug to inhibit LDL oxidation, interacted with lipoproteins in intact human plasma. In the present study, we compare the binding affinities of IBP to LDL and HDL (high-density lipoprotein) by (1)H NMR spectroscopy. When IBP is added into the HDL and LDL samples, the - N(+)(CH(3))(3) moieties of phosphatidylcholine (PC) and sphingomyelin (SM) in lipoprotein particles experience the chemical shift up-field drift. Intermolecular cross-peaks observed in NOESY spectra imply that there are direct interactions between ibuprofen and lipoproteins at both hydrophobic and hydrophilic (ionic) regions. These interactions are likely to be important in the solubility of ibuprofen into lipoprotein particles. Ibuprofen has higher impact on the PC and SM head group ( - N(+)(CH(3))(3)) and - (CH(2))(n) - group in HDL than that in LDL. This could be explained by either IBP has higher binding affinity to HDL than to LDL, or IBP induces orientation of the phospholipid head group at the surface of the lipoprotein particles.