Molecular insights into protein synthesis with proline residues

Proline is an amino acid with a unique cyclic structure that facilitates the folding of many proteins, but also impedes the rate of peptide bond formation by the ribosome. As a ribosome substrate, proline reacts markedly slower when compared with other amino acids both as a donor and as an acceptor of the nascent peptide. Furthermore, synthesis of peptides with consecutive proline residues triggers ribosome stalling. Here, we report crystal structures of the eukaryotic ribosome bound to analogs of mono‐ and diprolyl‐tRNAs. These structures provide a high‐resolution insight into unique properties of proline as a ribosome substrate. They show that the cyclic structure of proline residue prevents proline positioning in the amino acid binding pocket and affects the nascent peptide chain position in the ribosomal peptide exit tunnel. These observations extend current knowledge of the protein synthesis mechanism. They also revise an old dogma that amino acids bind the ribosomal active site in a uniform way by showing that proline has a binding mode distinct from other amino acids.     

Vascular permeability induced by VEGF family members in vivo: role of endogenous PAF and NO synthesis

We previously reported that vascular endothelial growth factor (VEGF) increases vascular permeability through the synthesis of endothelial platelet-activating factor (PAF), while others reported the contribution of nitric oxide (NO). Herein, we addressed the contribution of VEGF receptors and the role played by PAF and NO in VEGF-induced plasma protein extravasation. Using a modified Miles assay, intradermal injection in mice ears of VEGF-A(165), VEGF-A(121), and VEGF-C (1 microM) which activate VEGFR-2 (Flk-1) receptor increased vascular permeability, whereas a treatment with VEGFR-1 (Flt-1) analogs; PlGF and VEGF-B (1 microM) had no such effect. Pretreatment of mice with PAF receptor antagonist (LAU8080) or endothelial nitric oxide synthase (eNOS) inhibitor (L-NAME) abrogated protein extravasation mediated by VEGF-A(165). As opposed to PAF (0.01-1 microM), treatment with acetylcholine (ACh; up to 100 microM; inducer of NO synthesis) or sodium nitroprusside (SNP; up to 1 microM; NO donor) did not induce protein leakage. Simultaneous pretreatment of mice with eNOS and protein kinase A (PKA) inhibitors restored VEGF-A(165) vascular hyperpermeability suggesting that endogenous NO synthesis leads to PKA inhibition, which support maintenance of vascular integrity. Our data demonstrate that VEGF analogs increase vascular permeability through VEGFR-2 activation, and that both endogenous PAF and NO synthesis contribute to VEGF-A(165)-mediated vascular permeability. However, PAF but not NO directly increases vascular permeability per se, thereby, suggesting that PAF is a direct inflammatory mediator, whereas NO serves as a cofactor in VEGF-A(165) proinflammatory activities.     

小麦抗赤霉病品种NBS同源序列克隆与分析

根据二穗短柄草NBS-LRR类基因的保守序列设计同源引物,以小麦抗赤霉病品种苏麦3号、宁7840和望水白基因组DNA为模板,通过PCR扩增,得到43条序列,其中4条为非编码序列或结构域不完整;39条与植物抗病基因同源,其中的7条内部存在终止密码子,可能是假基因,经过比对分析,其余32条具有连续的开放阅读框和保守结构域,推导的氨基酸序列均具有Kinase-1a、Kinase-2和Kinase-3a及GLPL区等几个保守区,在GenBank中均能找到与之高度同源的其他物种的核酸序列,并且Kinase-2的最后一个氨基酸均为色氨酸(W),属于non-TIR类NBS基因。32条序列可分为4大类,它们之间核苷酸同源性为64%-98%,编码氨基酸同源性为22%-98%。根据序列分析随机设计5对不同基因特异性引物,并利用RT-PCR技术进行表达分析,结果表明,7-1、s-3、s-4和w-2均能表达,说明这些片段可能是功能性抗病基因的部分序列;7-13不表达,再次证明属于假基因。32条序列在之前未被报道过,这些RGA可以作为筛选赤霉病功能性抗病基因的候选序列。     

Structure-activity relationship of amiloride analogs as blockers of epithelial Na channels: II. Side-chain modifications

Summary The overall on- and off-rate constants for blockage of epithelial Na channels by amiloride analogs were estimated by noise analysis of the stationary Na current traversing frog skin epithelium. The (2-position) side chain structure of amiloride was varied in order to obtain structure/rate constant relationships. (1) Hydrophobic chain elongations (benzamil and related compounds of high blocking potency) increase the stability of the blocking complex (lowered off-rate), explained by attachment of the added phenyl moiety to a hydrophobic area near the site of side chain interaction with the channel protein. (2) Some other chain modifications show that the on-rate, which is smaller than a diffusion-limited rate, varies with side chain structure. In several cases this effect is not attributable to steric hindrance on encounter, and implies that the side chain interacts briefly with the channel protein (encounter complex) before the main blocking position of the molecule is attained. The encounter complex must be labile since the overall rate constants of blockage are not concentration-dependent. (3) In two cases, changes at the 2-position side chain and at other ring ligands, with known effects on the blocking rate constants, could be combined in one analog. The rate constants of blocking by the resulting compounds indicate that the structural changes have additive effects in terms of activation energies. (4) Along with other observations (voltage dependence of the rate constants and competition with the transported Na ion), these results suggest a blocking process of at least two steps. It appears that initially the 2-position side chain invades the outward-facing channel entrance, establishing a labile complex. Then the molecule is either released completely (no block) or the 6-ligand of the pyrazine ring gains access to its receptor counterpart, thus establishing the blocking complex, the lifetime of which is strongly determined by the electronegativity of the 6-ligand.     

Pituitary somatostatin receptors: dissociation at the pituitary level of receptor affinity and biological activity for selective somatostatin analogs

High affinity and saturable binding of [125I-Tyr11]somatostatin (SS) is described in membrane homogenates from a pituitary transplantable tumor (GH4C1) rich in somatotrophs (KD for SS = 0.67 nM; Bmax = 30 fmol/mg protein). Binding characteristics and pharmacology are similar to those measured on normal pituitary membranes. The potency of various SS analogs highly correlates with that measured in in vitro bioassay for growth hormone. This suggests that those GH4C1 membranes are a good model for SS receptors on somatotrophs. Interestingly however, analogs in which the Asn5 is deleted (Des-Asn5) or D-Ser replaces Ser13 show dissociated potencies between the various assays: [D-Ser13] analogs are more potent in pituitary than in GH4C1 membranes binding assay. Des-Asn5-modified analogs are much more potent in both pituitary binding assays than in the bioassay. This could reflect a multiplicity of SS receptor subtypes in pituitary.     

Approaches to the design of biochemical probes for positron emission tomography

Since the development of the 2-deoxy-D-glucose procedure by L. Sokoloff considerable advances have been made in the design of radiotracers for estimation of in-vivo biochemical parameters. Many of these advances are due to the development of positron emission tomography. As a result key biochemical processes can now be evaluated with newly developed positron-emitting labeled enzyme probes in man, in-vivo, allowing the study of a wide range of specific cellular processes in health and disease states.Special issue dedicated to Dr. Louis Sokoloff.     

An efficient synthesis of SK-658 and its analogs as potent histone deacetylase inhibitors

SK-658 is a potent histone deacetylase (HDAC) inhibitor that showed higher activity than SAHA due to the presence of extended hydrophobic group. We designed and synthesized thioester and SS-hybrid bearing SK-658 analogs as HDAC inhibitors. All the compounds were active in nano molar range and showed higher inhibitory activity than SAHA and SK-658. Among these, disulfide compounds showed the highest activity.     

Antimicrobial activity of human α-defensin 5 and its linear analogs: N-terminal fatty acylation results in enhanced antimicrobial activity of the linear analogs

Human α-defensin 5 (HD5) exhibits broad spectrum antimicrobial activity and plays an important role in mucosal immunity of the small intestine. Although there have been several studies, the structural requirements for activity and mechanism of bacterial killing is yet to be established unequivocally. In this study, we have investigated the antimicrobial activity of HD5 and linear analogs. Cysteine deletions attenuated the antibacterial activity considerably. Candidacidal activity was affected to a lesser extent. Fatty acid conjugated linear analogs showed antimicrobial activity comparable activity to HD5. Effective surface charge neutralization of bacteria was observed for HD5 as compared to the non-fatty acylated linear analogs. Our results show that HD5 and non-fatty acylated linear analogs enter the bacterial cytoplasm without causing damage to the bacterial inner membrane. Although fatty acylated peptides exhibited antimicrobial activity comparable to HD5, their mechanism of action involved permeabilization of the Escherichia coli inner membrane. HD5 and analogs had the ability to bind plasmid DNA. HD5 had greater binding affinity to plasmid DNA as compared to the analogs. The three dimensional structure of HD5 favors greater interaction with the bacterial cell surface and also with DNA. Antibacterial activity of HD5 involves entry into bacterial cytoplasm and binding to DNA which would result in shut down of the bacterial metabolism leading to cell death. We show how a moderately active linear peptide derived from the α-defensin HD5 can be engineered to enhance antimicrobial activity almost comparable to the native peptide.     

Short KR‐12 analogs designed from human cathelicidin LL‐37 possessing both antimicrobial and antiendotoxic activities without mammalian cell toxicity

KR‐12 (residues 18–29 of LL‐37) was known to be the smallest peptide of human cathelicidin LL‐37 possessing antimicrobial activity. In order to optimize α‐helical short antimicrobial peptides having both antimicrobial and antiendotoxic activities without mammalian cell toxicity, we designed and synthesized a series of KR‐12 analogs. Highest hydrophobic analogs KR‐12‐a5 and KR‐12‐a6 displayed greater inhibition of lipopolysaccharide (LPS)‐stimulated tumor necrosis factor‐α production and higher LPS‐binding activity. We have observed that antimicrobial activity is independent of charge, but LPS neutralization requires a balance of hydrophobicity and net positive charge. Among KR‐12 analogs, KR‐12‐a2, KR‐12‐a3 and KR‐12‐a4 showed much higher cell specificity for bacteria over erythrocytes and retained antiendotoxic activity, relative to parental LL‐37. KR‐12‐a5 displayed the strongest antiendotoxic activity but almost similar cell specificity as compared with LL‐37. Also, these KR‐12 analogs (KR‐12‐a2, KR‐12‐a3, KR‐12‐a4 and KR‐12‐a5) exhibited potent antimicrobial activity (minimal inhibitory concentration: 4 μM) against methicillin‐resistant Staphylococcus aureus. Taken together, these KR‐12 analogs have the potential for future development as a novel class of antimicrobial and anti‐inflammatory therapeutic agents. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.