Apical Scaffolding Protein NHERF2 Modulates the Localization of Alternatively Spliced Plasma Membrane Ca2+ Pump 2B Variants in Polarized Epithelial Cells

The membrane localization of the plasma membrane Ca²⁺-ATPase isoform 2 (PMCA2) in polarized cells is determined by alternative splicing; the PMCA2w/b splice variant shows apical localization, whereas the PMCA2z/b and PMCA2x/b variants are mostly basolateral. We previously reported that PMCA2b interacts with the PDZ protein Na⁺/H⁺ exchanger regulatory factor 2 (NHERF2), but the role of this interaction for the specific membrane localization of PMCA2 is not known. Here we show that co-expression of NHERF2 greatly enhanced the apical localization of GFP-tagged PMCA2w/b in polarized Madin-Darby canine kidney cells. GFP-PMCA2z/b was also redirected to the apical membrane by NHERF2, whereas GFP-PMCA2x/b remained exclusively basolateral. In the presence of NHERF2, GFP-PMCA2w/b co-localized with the actin-binding protein ezrin even after disruption of the actin cytoskeleton by cytochalasin D or latrunculin B. Surface biotinylation and fluorescence recovery after photobleaching experiments demonstrated that NHERF2-mediated anchorage to the actin cytoskeleton reduced internalization and lateral mobility of the pump. Our results show that the specific interaction with NHERF2 enhances the apical concentration of PMCA2w/b by anchoring the pump to the apical membrane cytoskeleton. The data also suggest that the x/b splice form of PMCA2 contains a dominant lateral targeting signal, whereas the targeting and localization of the z/b form are more flexible and not fully determined by intrinsic sequence features.     

植物基因打靶技术及其应用

本文对植物基因打靶技术的原理、操作程序、打靶效率的影响因素及其在植物中的应用现状进行了综述,并就如何有效的提高打靶效率提出了建议,同时对该技术在植物学研究领域中的应用前景进行了展望。     

Signal sequence recognition and protein targeting

Intracellular traffic is often controlled not by highways, but by handshakes and partner introductions within a cellular network. Recently determined structures suggest how signal sequences are recognized and how the GTP affinities of the signal recognition particle and its receptor are coupled to the targeting of ribosomes to translocational membrane pores. The structure of signal peptidase suggests how it releases functional proteins.     

Preclinical and Clinical Evidence for Efficient Opsonization of Poly(Ethyleneglycol)-Liposomes

Abstract

The clinical use of poly(ethyleneglycol)-coated liposomes (PEG-liposome) is becoming increasingly important. Here we summarize our recent preclinical and clinical evidence pointing to the possibility of efficient opsonization of intravenously injected PEG-liposomes under certain circumstances. Drastically enhanced blood clearance of PEG-liposomes was observed at low lipid dose and in case of multiple injection schemes. Further clinical evaluation of the observed “anti-stealth” phenomenon is indicated.     

In vivo phage display — A discovery tool in molecular biomedicine

In vivo phage display is a high-throughput method for identifying target ligands specific for different vascular beds. Targeting is possible due to the heterogeneous expression of receptors and other antigens in a particular vascular bed. Such expression is additionally influenced by the physiological or pathological status of the vasculature. In vivo phage display represents a technique that is usable in both, vascular mapping and targeted drug development. In this review, several important methodological aspects of in vivo phage display experiments are discussed. These include choosing an appropriate phage library, an appropriate animal model and the route of phage library administration. In addition, peptides or antibodies identified by in vivo phage display homing to specific types of vascular beds, including the altered vasculature present in several types of diseases are summarized. Still, confirmation in independent experiments and reproduction of identified sequences are needed for enhancing the clinical applicability of in vivo phage display research.     

Topogenesis and Homeostasis of Fatty Acyl-CoA Reductase 1

Peroxisomal fatty acyl-CoA reductase 1 (Far1) is essential for supplying fatty alcohols required for ether bond formation in ether glycerophospholipid synthesis. The stability of Far1 is regulated by a mechanism that is dependent on cellular plasmalogen levels. However, the membrane topology of Far1 and how Far1 is targeted to membranes remain largely unknown. Here, Far1 is shown to be a peroxisomal tail-anchored protein. The hydrophobic C terminus of Far1 binds to Pex19p, a cytosolic receptor harboring a C-terminal CAAX motif, which is responsible for the targeting of Far1 to peroxisomes. Far1, but not Far2, was preferentially degraded in response to the cellular level of plasmalogens. Experiments in which regions of Far1 or Far2 were replaced with the corresponding region of the other protein showed that the region flanking the transmembrane domain of Far1 is required for plasmalogen-dependent modulation of Far1 stability. Expression of Far1 increased plasmalogen synthesis in wild-type Chinese hamster ovary cells, strongly suggesting that Far1 is a rate-limiting enzyme for plasmalogen synthesis.     

Pretargeted radioimmunotherapy using 131I-labelled bivalent hapten-bearing peptides

Summary The advantages of bivalent hapten-bearing peptides for the detection of tumours pretargeted with bispecific antibodies have been demonstrated. This technology is now considered for radioimmunotherapy and bivalent haptens designed to target¹³¹I are needed. We thus synthesised a series of tyrosine-containing peptides bearing the histamine-hemisuccinate hapten. These molecules were tested for their ability to bind simultaneously two anti-hapten antibody molecules. One of these bivalent haptens, AG3.0, with a lysyl-d-tyrosyl-lysine connecting chain, was found to have optimal binding characteristics and was thus selected for further investigations. AG3.0 was shown to efficiently deliver radioactive iodine to human colorectal tumours grafted in nude mice using an anti-carcinoembryonic antigen×anti-histamine-hemisuccinate bispecific antibody. AG3.0 was also targeted to human B lymphoma cells pretargeted with a bispecific antibody specific for membrane IgM. In this system, bivalent ligands such as F(ab′)₂ or IgG are rapidly internalised and covalently linked radioactive iodine is released from target cells as a result of intracellular catabolism. With the pretargeted iodine-labelled bivalent hapten, a fivefold increase in the intracellular activity retention time as compared to¹²⁵I-labelled F(ab′)₂ and IgG was observed. The radiolabelled hapten did not undergo any degradation after internalisation. These results have been confirmed in vivo with an anti-BCL₁ IgM idiotype bispecific antibody and¹³¹I-labelled AG3.0. These reagents injected as a single 300 μCi dose, 7 days after inoculation of 10⁴ BCL₁ lymphoma cells in BALB/c mice, cured 14/16 of the animals and the treatment was well tolerated. Comparatively, the same dose of labelled IgG cured 13/16 of the mice but three mice died of haematologic toxicity. The same dose of labelled F(ab′)₂ or Fab′ was completely inefficient.¹³¹I-labelled bivalent haptens are now used in phase I radioimmunotherapy clinical trials.     

Retroviral display of urokinase-binding domain fused to amphotropic envelope protein

Tumors frequently express urokinase (uPA) receptor (uPAR). To investigate whether uPAR can efficiently target cancerous cells using amphotropic retroviral vectors, we generated a retrovirus displaying the amino-terminal fragment (ATF) of uPA as an N-terminal extension of viral envelope protein. We also made use of a "two-step strategy" by inserting a uPA cleavage site between the ATF moiety and the envelope. We measured the ability of ATF-bearing chimeric envelopes to infect huPAR-overexpressing Madin-Darby canine kidney (MDCK) and control MDCK II cells. The ATF-viruses infected both MDCK cell lines with an equivalent efficiency, suggesting that the chimeric viruses were not sequestered by uPAR and infect cells preferentially via the Pit-2 receptor. The addition of a uPA cleavage site increased the infection level of huPAR-MDCK cells by 2-fold when uPA was present in the infection medium. Surprisingly, ATF-env viruses infected huPAR-MDCK cells 5.5-fold more efficiently in the presence of exogenous uPA. This stimulatory effect of uPA on infection of huPAR-MDCK cells by the ATF-env virus was completely abolished by methyl-beta-cyclodextrin, suggesting that this effect involves the caveolar endocytosis pathway.     

应用改构的炭疽毒素作为靶向肿瘤细胞的药物递送系统及其效果评价

目的:通过改造炭疽毒素保护性抗原Protective Antigen (PA)及致死因子Lethal Factor (LF),尝试建立更加广谱的新型炭疽毒素靶向给药系统并对其递送效率进行定量评价.方法:采用基因工程手段,分别构建了3种改构的天然炭疽毒素保护性抗原PA及炭疽毒素的LF N端融合海肾荧光素酶(Luciferase)的LFn-linker-Luc的大肠杆菌重组表达体系.利用CCK-8法评价改构PA和LF共同作用肿瘤细胞后的细胞存活率;利用改构PA和LFn-linker-Luc与肿瘤细胞共孵育,通过测定细胞内荧光素酶活性,评价改构PA靶向肿瘤细胞的效果.结果:体外酶解实验证明构建的改构PA蛋白能够被正确地酶解成目的大小的片段;改构PA和LF共同作用肿瘤细胞能够显著降低细胞存活率;利用LFn-linker-Luc能够评价改构PA的靶向效率,PA蛋白的改构方式与其递送效率相关.结论:设计并改构的炭疽毒素药物递送系统,能够实现特异性靶向肿瘤细胞的效果,并具有更广谱的作用效果,为研制新型广谱抗肿瘤药物提供了新的思路和方法.