Enhancing native chemical ligation for challenging chemical protein syntheses

Native chemical ligation has enabled the chemical synthesis of proteins for a wide variety of applications (e.g., mirror-image proteins). However, inefficiencies of this chemoselective ligation in the context of large or otherwise challenging protein targets can limit the practical scope of chemical protein synthesis. In this review, we focus on recent developments aimed at enhancing and expanding native chemical ligation for challenging protein syntheses. Chemical auxiliaries, use of selenium chemistry, and templating all enable ligations at otherwise suboptimal junctions. The continuing development of these tools is making the chemical synthesis of large proteins increasingly accessible.     

贻贝棘尾虫(原生动物,纤毛门)镜像骈体自身各部移接的实验

棘尾虫是细胞表面具有特殊纤毛结构排列方式的复杂下毛类纤毛虫。其镜像骈体的两列纤毛结构几乎完全是左右对称排列 ,一个是正常方式排列的 ,另一个是相反方式排列。本文包括两项移接实验 :1)将棘尾虫口对镜像骈体一侧细胞的后半部分倒转 180°与另一侧细胞的前半部分对接 ;2 )将此种骈体一侧后半部分调转90°与另一侧细胞的前半部分对接。接后定时分期固定接块 ,以蛋白银染色法 ,观察其发育过程及结果。实验 1的发育结果为有的仍形成口对镜像骈体 ;有的成为末端对末端的镜像骈体。实验 2的发育结果为全部成为口对镜像骈体。两项实验结果均可用位置值假说解释 ,实验 1属于位置值插入的结果 ;实验 2为位置值添加的结果。为何实验Ⅰ有的发育成口对镜像骈体有的发育成末端对末端的镜像骈体 ,而实验 2却全部发育成口对镜像骈体呢 ?这是接块发育中是否发生对折的结果。Shietal.(1991)认为与预存的缘棘毛和是否具有完整表膜的面积大小有关系。接块在发育中是否发生对折与接块的伤口一侧面积大小和具完整表膜有关系。接块具完整表膜和缘棘毛的一侧由于新缘棘毛和口围带的发育生长 ,造成该侧的伸张 ,产生前后两接块向伤口一侧对折的压力 ,从而接块发生对折形成口对镜像骈体。我们认为还应有其它因素 :(1)与细胞     

Generation of deviation parameters for amino acid singlets, doublets and triplets from three-dimensional structures of proteins and its implications for secondary structure prediction from amino acid sequences

We present a new method, secondary structure prediction by deviation parameter (SSPDP) for predicting the secondary structure of proteins from amino acid sequence. Deviation parameters (DP) for amino acid singlets, doublets and triplets were computed with respect to secondary structural elements of proteins based on the dictionary of secondary structure prediction (DSSP)-generated secondary structure for 408 selected nonhomologous proteins. To the amino acid triplets which are not found in the selected dataset, a DP value of zero is assigned with respect to the secondary structural elements of proteins. The total number of parameters generated is 15,432, in the possible parameters of 25,260. Deviation parameter is complete with respect to amino acid singlets, doublets, and partially complete with respect to amino acid triplets. These generated parameters were used to predict secondary structural elements from amino acid sequence. The secondary structure predicted by our method (SSPDP) was compared with that of single sequence (NNPREDICT) and multiple sequence (PHD) methods. The average value of the percentage of prediction accuracy for αhelix by SSPDP, NNPREDICT and PHD methods was found to be 57%, 44% and 69% respectively for the proteins in the selected dataset. For Β-strand the prediction accuracy is found to be 69%, 21% and 53% respectively by SSPDP, NNPREDICT and PHD methods. This clearly indicates that the secondary structure prediction by our method is as good as PHD method but much better than NNPREDICT method.     

Understanding and controlling amyloid aggregation with chirality

Amyloid aggregation and human disease are inextricably linked. Examples include Alzheimer disease, Parkinson disease, and type II diabetes. While seminal advances on the mechanistic understanding of these diseases have been made over the last decades, controlling amyloid fibril formation still represents a challenge, and it is a subject of active research. In this regard, chiral modifications have increasingly been proved to offer a particularly well-suited approach toward accessing to previously unknown aggregation pathways and to provide with novel insights on the biological mechanisms of action of amyloidogenic peptides and proteins. Here, we summarize recent advances on how the use of mirror-image peptides/proteins and d-amino acid incorporations have helped modulate amyloid aggregation, offered new mechanistic tools to study cellular interactions, and allowed us to identify key positions within the peptide/protein sequence that influence amyloid fibril growth and toxicity.     

Comparison of Singlet and Doublet Paramecium tetraurelia: DNA Content,Protein Content and the Cell Cycle*

SYNOPSIS Doublet Paramecium tetraurelia contain either a single macronucleus which is substantially larger than that in a singlet cell, or 2 smaller macronuclei. Doublets have approximately twice the DNA content and twice the total protein content of singlets. The cell cycle of doublets is 164% as long as that of singlets, but the relative position of the macronuclear DNA synthesis period within the cell cycle is the same as in singlets. The DNA content of doublets is regulated so that differences in the number of macronuclei do not produce corresponding changes in DNA content; bimacronucleate doublets have only 27% more DNA than unimacronucleate doublets.     

Investigation of the inhibitory mechanism of apomorphine against MDM2–p53 interaction

Mirror-image screening using d-proteins is a powerful approach to provide mirror-image structures of chiral natural products for drug screening. During the course of our screening study for novel MDM2–p53 interaction inhibitors, we identified that NPD6878 (R-(?)-apomorphine) inhibited both the native l-MDM2–l-p53 interaction and the mirror-image d-MDM2–d-p53 interaction at equipotent doses. In addition, both enantiomers of apomorphine showed potent inhibitory activity against the native MDM2–p53 interaction. In this study, we investigated the inhibitory mechanism of both enantiomers of apomorphine against the MDM2–p53 interaction. Achiral oxoapomorphine, which was converted from chiral apomorphines under aerobic conditions, served as the reactive species to form a covalent bond at Cys77 of MDM2, leading to the inhibitory effect against the binding to p53.     

Mu-Opioid Receptor in the Nucleus Submedius: Involvement in Opioid-Induced Inhibition of Mirror-Image Allodynia in a Rat Model of Neuropathic Pain

The current study investigated the roles of various subtypes of opioid receptors expressed in the thalamic nucleus submedius (Sm) in inhibition of mirror-image allodynia induced by L5/L6 spinal nerve ligation in rats. Morphine was microinjected into the Sm, which produced a dose-dependent inhibition of mirror-image allodynia; this effect was antagonized by pretreatment with non-selective opioid receptor antagonist naloxone. Microinjections of endomorphin-1 (μ-receptor agonist), or [d-Ala², d-Leu⁵]-enkephalin (DADLE, δ-/μ-receptor agonist), also inhibited mirror-image allodynia, and these effects were blocked by the selective μ-receptor antagonist, β-funaltrexamine hydrochloride. The DADLE-induced inhibition, however, was not influenced by the δ-receptor antagonist naltrindole. The κ-receptor agonist, spiradoline mesylate salt, failed to alter the mirror-image allodynia. These results suggest that Sm opioid receptor signaling is involved in inhibition of mirror-image allodynia; this effect is mediated by μ- (but not δ- and κ-) opioid receptors in the rat model of neuropathic pain. Special issue article in honor of Dr. Ji-Sheng Han.     

Calcium and the role of motoneuronal doublets in skeletal muscle control

This work presents a novel structural model of skeletal muscle activation, providing a physiologically based account of frequency-dependent muscle responses like the catch-like effect. Numerous Ca²⁺ reservoirs within muscle fibers are considered, and a simplified analysis of the allocation of Ca²⁺ resources and the dynamics of calcium transport is proposed. The model correctly accounts for catch-like effects in slow and fast-twitch fibers during long-train stimulations and force–frequency relations in different muscle types. Results obtained from the model compare favorably to experiments showing that prolonged increases in force characteristic of the catch-like effect are not accompanied by sustained increases in free myoplasmic Ca²⁺. Also, in agreement with early experiments, the interspike interval in catch-inducing doublets is seen to be an important parameter for regulating the precise onset amplitude of the catch-like effect. This suggests that a plausible physiological function for the inclusion of doublets or the exclusion of individual spikes within a regular motor-neuronal spike-train is to rapidly bring skeletal muscles to predefined target forces according to prespecified motor programs in the central nervous system. This is a potentially very useful property directly mediated by the catch-like process modeled here. One further prediction of the model is that the slope of the frequency–tension profile of a given muscle is highly sensitive to changes in the efficiency and temporal characteristics of the dihydropyridine–ryanodine receptor complex. Interestingly, this is consistent with findings made on cardiac muscles, and might incidentally explain some instances of cardiac failure.     

Synthesis and structure-activity relationship of tyrosinase inhibiting novel bi-heterocyclic acetamides: Mechanistic insights through enzyme inhibition,kinetics and computational studies

The present research was designed for the selective synthesis of novel bi-heterocyclic acetamides, 9a-n, and their tyrosinase inhibition to overwhelm the problem of melanogenesis. The structures of newly synthesized compounds were confirmed by spectral techniques such as ¹H NMR, ¹³C NMR, and EI-MS along with elemental analysis. The inhibitory effects of these bi-heterocyclic acetamides (9a-n) were evaluated against tyrosinase and all these molecules were recognized as potent inhibitors relative to the standard used. The Kinetics mechanism was analyzed by Lineweaver-Burk plots which explored that compound, 9h, inhibited tyrosinase competitively by forming an enzyme-inhibitor complex. The inhibition constants K_i calculated from Dixon plots for this compound was 0.0027 µM. The computational study was coherent with the experimental records and these ligands exhibited good binding energy values (kcal/mol). The hemolytic analysis revealed their mild cytotoxicity towards red blood cell membranes and hence, these molecules can be pondered as nontoxic medicinal scaffolds for skin pigmentation and related disorders.     

A Mixed Mirror-image DNA/RNA Aptamer Inhibits Glucagon and Acutely Improves Glucose Tolerance in Models of Type 1 and Type 2 Diabetes

Excessive secretion of glucagon, a functional insulin antagonist, significantly contributes to hyperglycemia in type 1 and type 2 diabetes. Accordingly, immunoneutralization of glucagon or genetic deletion of the glucagon receptor improved glucose homeostasis in animal models of diabetes. Despite this strong evidence, agents that selectively interfere with endogenous glucagon have not been implemented in clinical practice yet. We report the discovery of mirror-image DNA-aptamers (Spiegelmer®) that bind and inhibit glucagon. The affinity of the best binding DNA oligonucleotide was remarkably increased (>25-fold) by the introduction of oxygen atoms at selected 2′-positions through deoxyribo- to ribonucleotide exchanges resulting in a mixed DNA/RNA-Spiegelmer (NOX-G15) that binds glucagon with a K_d of 3 nm. NOX-G15 shows no cross-reactivity with related peptides such as glucagon-like peptide-1, glucagon-like peptide-2, gastric-inhibitory peptide, and prepro-vasoactive intestinal peptide. In vitro, NOX-G15 inhibits glucagon-stimulated cAMP production in CHO cells overexpressing the human glucagon receptor with an IC₅₀ of 3.4 nm. A single injection of NOX-G15 ameliorated glucose excursions in intraperitoneal glucose tolerance tests in mice with streptozotocin-induced (type 1) diabetes and in a non-genetic mouse model of type 2 diabetes. In conclusion, the data suggest NOX-G15 as a therapeutic candidate with the potential to acutely attenuate hyperglycemia in type 1 and type 2 diabetes.