Surface plasmon resonance for high-throughput ligand screening of membrane-bound proteins

Technologies based on surface plasmon resonance (SPR) have allowed rapid, label-free characterization of protein-protein and protein-small molecule interactions. SPR has become the gold standard in industrial and academic settings, in which the interaction between a pair of soluble binding partners is characterized in detail or a library of molecules is screened for binding against a single soluble protein. In spite of these successes, SPR is only beginning to be adapted to the needs of membrane-bound proteins which are difficult to study in situ but represent promising targets for drug and biomarker development. Existing technologies, such as BIAcoreTM, have been adapted for membrane protein analysis by building supported lipid layers or capturing lipid vesicles on existing chips. Newer technologies, still in development, will allow membrane proteins to be presented in native or near-native formats. These include SPR nanopore arrays, in which lipid bilayers containing membrane proteins stably span small pores that are addressable from both sides of the bilayer. Here, we discuss current SPR instrumentation and the potential for SPR nanopore arrays to enable quantitative, high-throughput screening of G protein coupled receptor ligands and applications in basic cellular biology.     

Assessment of the effect of cyclic chalcone analogues on mitochondrial membrane and DNA

The cytotoxic and protective effects of selected synthetic chalcone analogues have been shown in previous studies. We studied their cytotoxic effect on the modification of mitochondrial membrane potential and on DNA. The first spectral information about the methoxy group as well as the dimethylamino substituent in E-2-arylmethylene-1-benzosuberones molecule was obtained by absorption and emission spectra. The cytotoxic effect of both cyclic chalcone analogues on DNA were detected by alkaline single-cell gel electrophoresis. Better fluorescent chalcone analogue E-2-(4′-dimethylamino-benzylidene)-1-benzosuberone was studied further in fresh isolated mitochondria. The decrease of rat liver mitochondria membrane potential (Δψ) was observed by fluorescence emission spectra. For the collapsing of mitochondrial potentials and as the negative control of mitochondrial function the CCCP uncoupler was used. The absorption maximum of the methoxy group was found at a shorter wavelength (λ = 335 nm) than that of the dimethylamino group (λ = 406 nm). The excitation spectra were very similar to the absorption spectra for both molecules but the emission spectra showed a better fluorescence for dimethylamino derivative. After the addition of E-2-(4′-dimethylamino-benzylidene)-1-benzosuberone to the intact mitochondria the decrease of mitochondrial membrane potential Δψ was observed by emisssion fluorescence spectra. Both cyclic chalcone analogues induced DNA damage, which was detected by alkaline comet assay. Mainly the apoptotic cells were detected, but necrotic cells were also present. Similarities in the percentages of DNA migration from the head were observed in both treatment groups. Both benzosuberones, with dimethylamino- and methoxy- substituent, were very active biologically, as shown by DNA results of the comet assay. Due to its better fluorescence properties, only the fluorophore with dimethylamino substituent was selected for further study of the function of rat liver mitochondria. Decline of mitochondrial function as well as mitochondrial DNA damage were evident between experimental and control groups.     

Direct binding of cholesterol to the amyloid precursor protein: An important interaction in lipid–Alzheimer's disease relationships?

It is generally believed that cholesterol homoeostasis in the brain is both linked to and impacted by Alzheimer's disease (AD). For example, elevated levels of cholesterol in neuronal plasma and endosome membranes appear to be a pro-amyloidogenic factor. The recent observation that the C-terminal transmembrane domain (C99, also known as the β-C-terminal fragment, or β-CTF) of the amyloid precursor protein (APP) specifically binds cholesterol helps to tie together previously loose ends in the web of our understanding of Alzheimer's–cholesterol relationships. In particular, binding of cholesterol to C99 appears to favor the amyloidogenic pathway in cells by promoting localization of C99 in lipid rafts. In turn, the products of this pathway—amyloid-β and the intracellular domain of the APP (AICD)—may down-regulate ApoE-mediated cholesterol uptake and cholesterol biosynthesis. If confirmed, this negative-feedback loop for membrane cholesterol levels has implications for understanding the function of the APP and for devising anti-amyloidogenic preventive strategies for AD.     

Using stacked generalization to predict membrane protein types based on pseudo-amino acid composition

Membrane proteins are vitally important for many biological processes and have become an attractive target for both basic research and drug design. Knowledge of membrane protein types often provides useful clues in deducing the functions of uncharacterized membrane proteins. With the unprecedented increasing of newly found protein sequences in the post-genomic era, it is highly demanded to develop an automated method for fast and accurately identifying the types of membrane proteins according to their amino acid sequences. Although quite a few identifiers have been developed in this regard through various approaches, such as covariant discriminant (CD), support vector machine (SVM), artificial neural network (ANN), and K-nearest neighbor (KNN), classifier the way they operate the identification is basically individual. As is well known, wise persons usually take into account the opinions from several experts rather than rely on only one when they are making critical decisions. Likewise, a sophisticated identifier should be trained by several different modes. In view of this, based on the frame of pseudo-amino acid that can incorporate a considerable amount of sequence-order effects, a novel approach called "stacked generalization" or "stacking" has been introduced. Unlike the "bagging" and "boosting" approaches which only combine the classifiers of a same type, the stacking approach can combine several different types of classifiers through a meta-classifier to maximize the generalization accuracy. The results thus obtained were very encouraging. It is anticipated that the stacking approach may also hold a high potential to improve the identification quality for, among many other protein attributes, subcellular location, enzyme family class, protease type, and protein-protein interaction type. The stacked generalization classifier is available as a web-server named "SG-MPt_Pred" at: http://202.120.37.186/bioinf/wangsq/service.htm.     

Chemical proteomic and bioinformatic strategies for the identification and quantification of vascular antigens in cancer

One avenue towards the development of more selective anti-cancer drugs consists in the targeted delivery of bioactive molecules to the tumor environment by means of binding molecules specific to tumor-associated markers. In this context, the targeted delivery of therapeutic agents to newly-formed blood vessels (“vascular targeting”) is particularly attractive, because of the dependence of tumors on new blood vessels to sustain growth and invasion, and because of the accessibility of neo-vascular structures for therapeutic agents injected intravenously. Ligand-based vascular targeting strategies crucially rely on good-quality vascular tumor markers. Here we describe a number of established technologies for the enrichment of accessible vascular proteins based on the isolation of glycoproteins, the in vivo coating of accessible cell surfaces with colloidal silica and the in vivo perfusion with reactive ester derivatives of biotin. Label-free as well as isotopic labeling based strategies for the subsequent MS-based protein quantification are outlined. Finally, bioinformatic workflows for protein quantification are depicted aiming at assisting in the evaluation of appropriate strategies for individual projects. This review gives an overview of current chemical proteomic strategies for the enrichment and quantification of the accessible vascular proteome and helps in selecting bioinformatic strategies for data analysis and validation.     

Phospholipase Dδ is Involved in Wounding Induced Phosphatidic Acid Formation in Arabidopsis

Phosphalipase D (PLD) hydrolyzes phospholipids into phosphatidic acid (PA). PLDα1 and δ are the two most abundant members of the 12 member PLD family in Arabidopsis. PLDα1 has been demonstrated having role in the wounding induced PA signalling. However, whether and how PLDδ is involved in wounding induced PA formation remained unclear. In the present study, the membrane lipids response to wounding was profiled in Wassilewskija (WS) and PLDδ knockout mutant (PLDδ KO) of Arabidopsis. The levels of most lipids, including monogalactosyldiacylglycerol, digalactosyldiacylglycerol, phosphatidylcholine and phosphatidylglycerol had decreased rapidly within 30min after wounding in the two Arabidopsis genotypes. In contrast, the level of PA increased sharply and significantly 30min after wounding. It continued to increase until peaking at 1h post wounding in WS and 3h post wounding in PLDδ KO, and then decreased. The PA levels were similar in the two genotypes in untreated leaves and in leaves 6h after wounding. However, these levels were lower in PLDδ KO than in WS from 30min to 3h post wounding. The significant difference of PA level between the two genotypes occurred 30min after wounding, when it was about 20% lower in PLDδ KO than in WS. These results show that PLDδ is involved in wounding induced PA formation in Arabidopsis, but its absence induces PA response later and with less intensity than PLDα1.     

High-resolution Native-PAGE for membrane proteins capable of fluorescence detection and hydrodynamic state evaluation

An improved native polyacrylamide gel electrophoresis (PAGE) method capable of evaluating the hydrodynamic states of membrane proteins and allowing in-gel fluorescence detection was established. In this method, bis(alkyl) sulfosuccinate is used to provide negative charges for detergent-solubilized membrane proteins to facilitate proper electrophoretic migration without disturbing their native hydrodynamic states. The method achieved high-resolution electrophoretic separation, in good agreement with the elution profiles obtained by size exclusion chromatography. The applicability of in-gel fluorescence detection for tagged green fluorescent protein (GFP) facilitates the analysis of samples without any purification. This method might serve as a general analytical technique for assessing the folding, oligomerization, and protein complex formation of membrane proteins.     

Direct quantification of mitochondrial DNA and its 4.9-kb common deletion without DNA purification

We studied the extraction and analysis of integral membrane proteins possessing hydrophobic and hydrophilic domains and found that a nonionic detergent called MEGA-10, used in lysis buffers, had a superior extraction effect compared to most conventional detergents. A sodium dodecyl sulfate (SDS) concentration of >0.4% (w/v) in the sample buffer was crucial for those proteins to be clearly analyzed by electrophoresis and Western blotting. Furthermore, MEGA-10 had the tendency to maximally extract proteins around its critical micelle concentration (CMC) of 0.24% (w/v). These solutions can greatly assist functional investigations of membrane proteins in the proteomics era.     

Synthesis and in situ insertion of a site-specific fluorescently labeled membrane protein into cell-sized liposomes

The integral membrane protein bacteriorhodopsin, containing a fluorescent amino acid at a specific position, was synthesized in the presence of hydrated lipid films using an in vitro translation system expanded with a four-base codon/anticodon pair. Cell-sized liposomes with the labeled protein inserted into the liposome membranes were generated after the translation reaction. This study also demonstrated that this labeling method could be used to analyze the dynamic properties of membrane proteins in situ by fluorescence correlation spectroscopy.     

Cellular levels of heme affect the activity of dimeric glutamyl-tRNA reductase

Prestin, a multipass transmembrane protein whose N- and C-termini are localized to the cytoplasm, must be trafficked to the plasma membrane to fulfill its cellular function as a molecular motor. One challenge in studying prestin sequence-function relationships within living cells is separating the effects of amino acid substitutions on prestin trafficking, plasma membrane localization and function. To develop an approach for directly assessing prestin levels at the plasma membrane, we have investigated whether fusion of prestin to a single pass transmembrane protein results in a functional fusion protein with a surface-exposed N-terminal tag that can be detected in living cells. We find that fusion of the biotin-acceptor peptide (BAP) and transmembrane domain of the platelet-derived growth factor receptor (PDGFR) to the N-terminus of prestin-GFP yields a membrane protein that can be metabolically-labeled with biotin, trafficked to the plasma membrane, and selectively detected at the plasma membrane using fluorescently-tagged streptavidin. Furthermore, we show that the addition of a surface detectable tag and a single-pass transmembrane domain to prestin does not disrupt its voltage-sensitive activity.