A flexible docking approach for prediction of T cell receptor–peptide–MHC complexes

T cell receptors (TCRs) are immune proteins that specifically bind to antigenic molecules, which are often foreign peptides presented by major histocompatibility complex proteins (pMHCs), playing a key role in the cellular immune response. To advance our understanding and modeling of this dynamic immunological event, we assembled a protein–protein docking benchmark consisting of 20 structures of crystallized TCR/pMHC complexes for which unbound structures exist for both TCR and pMHC. We used our benchmark to compare predictive performance using several flexible and rigid backbone TCR/pMHC docking protocols. Our flexible TCR docking algorithm, TCRFlexDock, improved predictive success over the fixed backbone protocol, leading to near‐native predictions for 80% of the TCR/pMHC cases among the top 10 models, and 100% of the cases in the top 30 models. We then applied TCRFlexDock to predict the two distinct docking modes recently described for a single TCR bound to two different antigens, and tested several protein modeling scoring functions for prediction of TCR/pMHC binding affinities. This algorithm and benchmark should enable future efforts to predict, and design of uncharacterized TCR/pMHC complexes.     

温度对中型指环虫产卵和孵化的影响

指环虫病是严重影响鱼类养殖的寄生虫病.为了有效控制指环虫病,实验研究了寄生在金鱼(Carassius auratus)鳃部的中型指环虫(Dactylogyrus intermedius)卵、纤毛幼虫的形态,以及在离体条件下温度对其产卵和孵化的影响.成熟的中型指环虫虫卵大部分为梨形,长30 μm左右,后端有一个卵柄.纤毛幼虫呈圆筒状,两端稍尖,眼点两对;后吸盘具有若干对小锚钩;在前部、中部和尾部分别具有一圈纤毛.实验研究了4℃、10℃、22℃、30℃和35℃条件下中型指环虫的产卵和孵化情况,在4℃条件下,中型指环虫基本不产卵也不孵化;在其他4个温度条件下,产卵量随着温度的升高而增加,其平均产卵量分别为3.30、4.10、4.13和4.24枚/虫.统计结果显示:在35℃条件下的产卵量明显高于10℃(P＜0.05),其他温度条件下的平均产卵量没有显著性差异.中型指环虫的产卵速率随着温度的升高而加快,产卵维持的时间分别为4d、23h、15h和llh.孵化率在22℃时最高,为72.7％,在30℃和35℃的孵化率为50％左右,卡方检验显示:4种温度下的孵化率之间没有显著性差异p＞0.05);随着温度的升高,孵化速率逐渐加快,而孵化时间和纤毛幼虫的存活时间则缩短,平均孵化时间分别为24d、3d、42h和26h,纤毛幼虫的最长存活时间分别为4d、3d、56h和34h.结果显示,当水温为22℃时,中型指环虫的产卵数量和纤毛幼虫的存活时间都比较高,且孵化率最高,表明该温度条件较适合中型指环虫的种群增长.     

Endoplasmic reticulum-associated degradation of a degron-containing polytopic membrane protein

Abstract

The presence of two basic amino acids strategically located within a single spanning transmembrane region has previously been shown to act as a signal for the endoplasmic reticulum associated degradation (ERAD) of several polypeptides. In contrast, the functionality of this degron motif within the context of a polytopic membrane protein has not been established. Using opsin as a model system, we have investigated the consequences of inserting the degron motif in the first of its seven transmembrane (TM) spans. Whilst these basic residue reduce the binding of the targeting factor, signal recognition particle, to the first TM span, this has no effect on membrane integration in vitro or in vivo. This most likely reflects the presence of multiple TM spans that can act as targeting signals within in the nascent opsin chain. We find that the degron motif leads to the efficient retention of mutant opsin chains at the endoplasmic reticulum. The mutant opsin polypeptides are degraded via a proteasomal pathway that involves the actions of the E3 ubiquitin ligase HRD1. In contrast, wild-type opsin remains stable for a prolonged period even when artificially accumulated at the endoplasmic reticulum. We conclude that a single dibasic degron motif is sufficient to initiate both the ER retention and subsequent degradation of ospin via an ERAD pathway.     

Foreword: Protein acylation and microdomains in membrane function

The Signal Recognition Particle (SRP) plays a critical role in the sorting of nascent secretory and membrane proteins. Remarkably, this function has been conserved from bacteria, where SRP delivers proteins to the inner membrane, through to eukaryotes, where SRP is required for targeting of proteins to the endoplasmic reticulum. This review focuses on present understanding of SRP structure and function and the relationship between the two. Furthermore, the similarities and differences in the structure, function and cellular role of SRP in bacteria, chloroplasts, fungi and mammals will be stressed.     

CHF5074 restores visual memory ability and pre‐synaptic cortical acetylcholine release in pre‐plaque Tg2576 mice

CHF5074, a new microglial modulator, attenuates memory deficit in Alzheimer's disease transgenic mice. In this study, the effect of an acute or subacute CHF5074 treatment on in vivo novel object recognition test and on [³H]Acetylcholine (ACh) and GABA release in pre‐plaque (7‐month‐old) Tg2576 mice have been compared with those induced by the γ‐secretase inhibitor LY450139 (semagacestat). Vehicle‐treated Tg2576 mice displayed an impairment of recognition memory compared with wild‐type animals. This impairment was recovered in transgenic animals acutely treated with CHF5074 (30 mg/kg), while LY450139 (1, 3, 10 mg/kg) was ineffective. In frontal cortex synaptosomes from vehicle‐treated Tg2576 mice, K⁺‐evoked [³H]ACh release was lower than that measured in wild‐type mice. This reduction was absent in transgenic animals subacutely treated with CHF5074 (30 mg/kg daily for 8 days), while it was slightly, not significantly, amplified by LY450139 (3 mg/kg daily for 8 days). There were no differences between the groups on spontaneous [³H]ACh release as well as spontaneous and K⁺‐evoked GABA release. These results suggest that CHF5074 has beneficial effects on visual memory and cortical cholinergic dysfunctions in pre‐plaque Tg2576 mice. Together with previous findings, these data suggest that CHF5074 could be a possible candidate for early Alzheimer's disease therapeutic regimens.     

Binding orientation and specificity of calmodulin to rat olfactory cyclic nucleotide-gated ion channel

Calmodulin (CaM), the primary intracellular Ca²⁺ receptor, regulates a large number of key enzymes and controls a wide spectrum of important biological responses. Recognition between CaM and its target sequence in rat olfactory cyclic nucleotide-gated ion channel (OLFp) was investigated by circular dichroism (CD), fluorescence, and NMR spectroscopy. Fluorescence data showed the OLFp tightly bound to CaM with a dissociation constant of 12?nM in a 1:1 stoichiometry. Far-UV CD data showed that approximately 60% of OLFp residues formed α-helical structures when associated with CaM. NMR data showed that most of the ¹⁵N–¹H HSQC cross-peaks of the ¹⁵N-labeled CaM not only shifted but also split into two sets of peaks upon association with the OLFp. Our data indicated that the two distinct CaM/OLFp complexes existed simultaneously with stable structures that were not interexchangeable within the NMR time scale. In light of the palindromic sequence of OLFp (FQRIVRLVGVIRDW) for CaM targeting, we proposed that the helical OLFp with C₂ symmetry may bind to CaM in two orientations. This hypothesis is supported by the observation that only one set of ¹⁵N–¹H HSQC cross-peaks of the ¹⁵N-labeled CaM was detected upon association with OLFp-M13 chimeric peptide (OLFMp), a mutated OLFp lacking the palindromic feature. The binding specificity of OLFMp to CaM was restored when the palindromic feature was destroyed. Binding modes of CaM/OLFp and CaM/OLFMp simulated by molecular docking were in accord with their distinct patterns observed in HSQC spectra. Our studies suggest that the palindromic residues in OLFp are crucial for the orientation-specific recognition by CaM.     

Recent progresses in gene delivery-based bone tissue engineering

Gene therapy has converged with bone engineering over the past decade, by which a variety of therapeutic genes have been delivered to stimulate bone repair. These genes can be administered via in vivo or ex vivo approach using either viral or nonviral vectors. This article reviews the fundamental aspects and recent progresses in the gene therapy-based bone engineering, with emphasis on the new genes, viral vectors and gene delivery approaches.