Mechanisms of cellular communication through intercellular protein transfer

In a multicellular system, cellular communication is a must for orchestration and coordination of cellular events. Advent of the latest analytical and imaging tools has allowed us to enhance our understanding of the intercellular communication. An intercellular exchange of proteins or intact membrane patches is a ubiquitous phenomenon, and has been the subject of renewed interest, particularly in the context of immune cells. Recent evidence implicates that intercellular protein transfers, including trogocytosis is an important mechanism of the immune system to modulate immune responses and transferred proteins can also contribute to pathology. It has been demonstrated that intercellular protein transfer can be through the internalization/pathway, dissociation-associated pathway, uptake of exosomes and membrane nanotube formations. Exchange of membrane molecules/antigens between immune cells has been observed for a long time, but the mechanisms and functional consequences of these transfers remain unclear. In this review, we will discuss the important findings concerning intercellular protein transfers, possible mechanisms and highlight their physiological relevance to the immune system, with special reference to T cells such as the stimulatory or suppressive immune responses derived from T cells with acquired dendritic cell membrane molecules.     

Serotonin receptor 2A gene moderates the effect of childhood maternal nurturance on adulthood social attachment

The ability to form and maintain attachment relations with other people is crucial for mental health and well-being. The origins of attachment behaviors are often assumed to be in early experiences with other people, especially with primary caregivers. Preliminary evidence suggests that serotonergic system may be involved in attachment behaviors. We examined whether the T102C variant of the serotonin receptor 2A gene moderates the effect of childhood maternal nurturance on social attachment in adulthood. The participants were 1070 women and men from the Young Finns Study with 27-year follow-up and two measurement times for the outcomes (n = 1836 person observations). Mothers reported their relationship quality with their children (participants) in childhood or adolescence. Social attachment was assessed by participant's self-reports on two measures (reward dependence scale of the Temperament and Character Inventory and the Relationship Questionnaire). High childhood maternal nurturance predicted high reward dependence and low avoidant attachment in carriers of the T/T genotype but not in the T/C or C/C genotype groups, while low maternal nurturance was associated with low reward dependence and high avoidant attachment in T/T genotype carriers but not in C allele carriers. Our result suggests that T/T genotype carriers were more influenced by their childhood nurturing environment, than their C allele carrying counterparts, thus providing evidence for differential susceptibility to childhood nurturing environment associated with the HTR2A gene.     

Phytosterols and phytosterolemia: gene–diet interactions

Phytosterol intake is recommended as an adjunctive therapy for hypercholesterolemia, and plant sterols/stanols can reduce cholesterol absorption at the intestinal lumen through the Niemann-Pick C1 Like 1 (NPC1L1) transporter pathway by competitive solubilization in mixed micelles. Phytosterol absorption is of less magnitude than cholesterol and is preferably secreted in the intestinal lumen by ABCG5/G8 transporters. Therefore, plasma levels of plant sterols/stanols are negligible compared with cholesterol, under an ordinary diet. The mechanisms of cholesterol and plant sterols absorption and the whole-body pool of sterols are discussed in this chapter. There is controversy about treatment with statins inducing further increase in plasma non-cholesterol sterols raising concerns about the safety of supplementation of plant sterols to such drugs. In addition, increase in plant sterols has also been reported upon consumption of plant sterol-enriched foods, regardless of other treatments. Rare mutations on ABCG5/G8 transporters affecting cholesterol/non-cholesterol extrusion, causing sitosterolemia with xanthomas and premature atheroslerotic disease are now known, and cholesterol/plant sterols absorption inhibitor, ezetimibe, emerges as the drug that reduces phytosterolemia and promotes xanthoma regression. On the other hand, common polymorphisms affecting the NPC1L1 transporter can interfere with the action of ezetimibe. Gene-diet interactions participate in this intricate network modulating the expression of genetic variants on specific phenotypes and can also affect the individual response to the hypolipidemic treatment. These very interesting aspects promoted a great deal of research in the field.     

Molecular modeling study of the opioid receptor interactions with series of cyclic deltorphin analogues

In this study, ten tetra‐ and heptapeptide analogues of deltorphin containing the urea bridges between residues 2 and 4 have been docked into the δ‐ and µ‐opioid receptors to explain their different biological activities. The important factors explaining particular ligand activity such as free energy of binding, conformation of the ligand, its location inside the binding pocket as well as the number and strength of the receptor–ligand interactions have been discussed. Several different binding modes for investigated ligands have been proposed. It appears that the binding site is not identical even for very similar ligands. Results of this study help to explain the differences in biological activity of the deltorphin analogues, their interaction with the opioid receptors at the molecular level and support designing a new generation of potent opioid drugs with improved selectivity. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Analysis of the interacting surface of maurotoxin with the voltage‐gated Shaker B K+ channel

Maurotoxin (MTX) is a 34‐residue toxin that was isolated initially from the venom of the scorpion Scorpio maurus palmatus. Unlike the other toxins of the α‐KTx6 family (Pi1, Pi4, Pi7, and HsTx1), MTX exhibits a unique disulfide bridge organization of the type C₁? C₅, C₂? C₆, C₃? C₄, and C₇? C₈ (instead of the conventional C₁? C₅, C₂? C₆, C₃? C₇, and C₄? C₈, herein referred to as Pi1‐like) that does not prevent its folding along the classic α/β scaffold of scorpion toxins. MTX_Pi1 is an MTX variant with a conventional pattern of disulfide bridging without any primary structure alteration of the toxin. Here, using MTX and/or MTX_Pi1 as models, we investigated how the type of folding influences toxin recognition of the Shaker B potassium channel. Amino acid residues of MTX that were studied for Shaker B recognition were selected on the basis of their homologous position in charybdotoxin, a three disulfide‐bridged scorpion toxin also active on this channel type. These residues favored either an MTX‐ or MTX_Pi1‐like folding. Our data indicate clearly that Lys²³ and Tyr³² (two out of ten amino acid residues studied) are the most important residues for Shaker B channel blockage by MTX. For activity on SKCa channels, the same amino acid residues also affect, directly or indirectly, the recognition of SK channels. The molecular modeling technique and computed docking indicate the existence of a correlation between the half cystine pairings of the mutated analogs and their activity on the Shaker B K⁺ channel. Overall, mutations in MTX could, or could not, change the reorganization of disulfide bridges of this molecule without affecting its α/β scaffold. However, changing of the peptide backbone (cross linking disulfide bridges from MTX‐like type vs MTX_Pi1‐like type) appears to have less impact on the molecule activity than mutation of certain key amino acids such as Lys²³ and Tyr³² in this toxin. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

乳腺生物反应器表达的重组人乳清白蛋白的高效分离纯化

利用乳腺生物反应器高效地表达重组人乳清白蛋白,但是目标产物分离纯化的难度较大。通过分子模拟计算比较待分离原料中主要蛋白组分的物化性质,包括表面电势和表面疏水性,在此基础上设计了高分辨率、快速的分离纯化工艺。通过硫酸铵沉淀的正交试验条件优化,有效地去除了干扰层析精制过程的IgG杂质,提高了后续疏水层析的稳定性,从而成功地分离开目标蛋白及与其同源的牛乳清白蛋白杂质,得到纯度>95%的rHLA纯品,工艺回收率48.6%。乳糖合成活性和圆二色谱检测结果表明,纯化rHLA具有调节β-1,4-半乳糖苷转移酶活性和天然的空间结构。     

A Calcium Bioluminescence Assay for Functional Analysis of Mosquito (Aedes aegypti) and Tick (Rhipicephalus microplus) G Protein-coupled Receptors

Arthropod hormone receptors are potential targets for novel pesticides as they regulate many essential physiological and behavioral processes. The majority of them belong to the superfamily of G protein-coupled receptors (GPCRs). We have focused on characterizing arthropod kinin receptors from the tick and mosquito. Arthropod kinins are multifunctional neuropeptides with myotropic, diuretic, and neurotransmitter function. Here, a method for systematic analyses of structure-activity relationships of insect kinins on two heterologous kinin receptor-expressing systems is described. We provide important information relevant to the development of biostable kinin analogs with the potential to disrupt the diuretic, myotropic, and/or digestive processes in ticks and mosquitoes.The kinin receptors from the southern cattle tick, Boophilus microplus (Canestrini), and the mosquito Aedes aegypti (Linnaeus), were stably expressed in the mammalian cell line CHO-K1. Functional analyses of these receptors were completed using a calcium bioluminescence plate assay that measures intracellular bioluminescence to determine cytoplasmic calcium levels upon peptide application to these recombinant cells. This method takes advantage of the aequorin protein, a photoprotein isolated from luminescent jellyfish. We transiently transfected the aequorin plasmid (mtAEQ/pcDNA1) in cell lines that stably expressed the kinin receptors. These cells were then treated with the cofactor coelenterazine, which complexes with intracellular aequorin. This bond breaks in the presence of calcium, emitting luminescence levels indicative of the calcium concentration. As the kinin receptor signals through the release of intracellular calcium, the intensity of the signal is related to the potency of the peptide.This protocol is a synthesis of several previously described protocols with modifications; it presents step-by-step instructions for the stable expression of GPCRs in a mammalian cell line through functional plate assays (Staubly et al., 2002 and Stables et al., 1997). Using this methodology, we were able to establish stable cell lines expressing the mosquito and the tick kinin receptors, compare the potency of three mosquito kinins, identify critical amino acid positions for the ligand-receptor interaction, and perform semi-throughput screening of a peptide library. Because insect kinins are susceptible to fast enzymatic degradation by endogenous peptidases, they are severely limited in use as tools for pest control or endocrinological studies. Therefore, we also tested kinin analogs containing amino isobutyric acid (Aib) to enhance their potency and biostability. This peptidase-resistant analog represents an important lead in the development of biostable insect kinin analogs and may aid in the development of neuropeptide-based arthropod control strategies.