GPCR-styrene maleic acid lipid particles (GPCR-SMALPs):their nature and potential

G-protein-coupled receptors (GPCRs) form the largest class of membrane proteins and are an important target for therapeutic drugs. These receptors are highly dynamic proteins sampling a range of conformational states in order to fulfil their complex signalling roles. In order to fully understand GPCR signalling mechanisms it is necessary to extract the receptor protein out of the plasma membrane. Historically this has universally required detergents which inadvertently strip away the annulus of lipid in close association with the receptor and disrupt lateral pressure exerted by the bilayer. Detergent-solubilized GPCRs are very unstable which presents a serious hurdle to characterization by biophysical methods. A range of strategies have been developed to ameliorate the detrimental effect of removing the receptor from the membrane including amphipols and reconstitution into nanodics stabilized by membrane scaffolding proteins (MSPs) but they all require exposure to detergent. Poly(styrene-co-maleic acid) (SMA) incorporates into membranes and spontaneously forms nanoscale poly(styrene-co-maleic acid) lipid particles (SMALPs), effectively acting like a 'molecular pastry cutter' to 'solubilize' GPCRs in the complete absence of detergent at any stage and with preservation of the native annular lipid throughout the process. GPCR-SMALPs have similar pharmacological properties to membrane-bound receptor, exhibit enhanced stability compared with detergent-solubilized receptors and being non-proteinaceous in nature, are fully compatible with downstream biophysical analysis of the encapsulated GPCR

Differences in SMA-like polymer architecture dictate the conformational changes exhibited by the membrane protein rhodopsin encapsulated in lipid nano-particles

Membrane proteins are of fundamental importance to cellular processes and nano-encapsulation strategies that preserve their native lipid bilayer environment are particularly attractive for studying and exploiting these proteins. Poly(styrene-co-maleic acid) (SMA) and related polymers poly(styrene-co-(N-(3-N′,N′-dimethylaminopropyl)maleimide)) (SMI) and poly(diisobutylene-alt-maleic acid) (DIBMA) have revolutionised the study of membrane proteins by spontaneously solubilising membrane proteins direct from cell membranes within nanoscale discs of native bilayer called SMA lipid particles (SMALPs), SMILPs and DIBMALPs respectively. This systematic study shows for the first time, that conformational changes of the encapsulated protein are dictated by the solubilising polymer. The photoactivation pathway of rhodopsin (Rho), a G-protein-coupled receptor (GPCR), comprises structurally-defined intermediates with characteristic absorbance spectra that revealed conformational restrictions with styrene-containing SMA and SMI, so that photoactivation proceeded only as far as metarhodopsin-I, absorbing at 478 nm, in a SMALP or SMILP. In contrast, full attainment of metarhodopsin-II, absorbing at 382 nm, was observed in a DIBMALP. Consequently, different intermediate states of Rho could be generated readily by simply employing different SMA-like polymers. Dynamic light-scattering and analytical ultracentrifugation revealed differences in size and thermostability between SMALP, SMILP and DIBMALP. Moreover, encapsulated Rho exhibited different stability in a SMALP, SMILP or DIBMALP. Overall, we establish that SMA, SMI and DIBMA constitute a ‘toolkit’ of solubilising polymers, so that selection of the appropriate solubilising polymer provides a spectrum of useful attributes for studying membrane proteins

Blockade of advanced glycation end product formation attenuates bleomycin-induced pulmonary fibrosis in rats

<p>Abstract</p> <p>Background</p> <p>Advanced glycation end products (AGEs) have been proposed to be involved in pulmonary fibrosis, but its role in this process has not been fully understood. To investigate the role of AGE formation in pulmonary fibrosis, we used a bleomycin (BLM)-stimulated rat model treated with aminoguanidine (AG), a crosslink inhibitor of AGE formation.</p> <p>Methods</p> <p>Rats were intratracheally instilled with BLM (5 mg/kg) and orally administered with AG (40, 80, 120 mg/kg) once daily for two weeks. AGEs level in lung tissue was determined by ELISA and pulmonary fibrosis was evaluated by Ashcroft score and hydroxyproline assay. The expression of heat shock protein 47 (HSP47), a collagen specific molecular chaperone, was measured with RT-PCR and Western blot. Moreover, TGFβ1 and its downstream Smad proteins were analyzed by Western blot.</p> <p>Results</p> <p>AGEs level in rat lungs, as well as lung hydroxyproline content and Ashcroft score, was significantly enhanced by BLM stimulation, which was abrogated by AG treatment. BLM significantly increased the expression of HSP47 mRNA and protein in lung tissues, and AG treatment markedly decreased BLM-induced HSP47 expression in a dose-dependent manner (p < 0.05). In addition, AG dose-dependently downregulated BLM-stimulated overexpressions of TGFβ1, phosphorylated (p)-Smad2 and p-Smad3 protein in lung tissues.</p> <p>Conclusion</p> <p>These findings suggest AGE formation may participate in the process of BLM-induced pulmonary fibrosis, and blockade of AGE formation by AG treatment attenuates BLM-induced pulmonary fibrosis in rats, which is implicated in inhibition of HSP47 expression and TGFβ/Smads signaling.</p

Role of liposome and peptide in the synergistic enhancement of transfection with a lipopolyplex vector

Lipopolyplexes are of widespread interest for gene therapy due to their multifunctionality and high transfection efficiencies. Here we compared the biological and biophysical properties of a lipopolyplex formulation with its lipoplex and polyplex equivalents to assess the role of the lipid and peptide components in the formation and function of the lipopolyplex formulation. We show that peptide efficiently packaged plasmid DNA forming spherical, highly cationic nanocomplexes that are taken up efficiently by cells. However, transgene expression was poor, most likely due to endosomal degradation since the polyplex lacks membrane trafficking properties. In addition the strong peptide-DNA interaction may prevent plasmid release from the complex and so limit plasmid DNA availability. Lipid/DNA lipoplexes, on the other hand, produced aggregated masses that showed poorer cellular uptake than the polyplex but contrastingly greater levels of transgene expression. This may be due to the greater ability of lipoplexes relative to polyplexes to promote endosomal escape. Lipopolyplex formulations formed spherical, cationic nanocomplexes with efficient cellular uptake and significantly enhanced transfection efficiency. The lipopolyplexes combined the optimal features of lipoplexes and polyplexes showing optimal cell uptake, endosomal escape and availability of plasmid for transcription, thus explaining the synergistic increase in transfection efficiency

Direct detection and measurement of wall shear stress using a filamentous bio-nanoparticle

The wall shear stress (WSS) that a moving fluid exerts on a surface affects many processes including those relating to vascular function. WSS plays an important role in normal physiology (e.g. angiogenesis) and affects the microvasculature's primary function of molecular transport. Points of fluctuating WSS show abnormalities in a number of diseases; however, there is no established technique for measuring WSS directly in physiological systems. All current methods rely on estimates obtained from measured velocity gradients in bulk flow data. In this work, we report a nanosensor that can directly measure WSS in microfluidic chambers with sub-micron spatial resolution by using a specific type of virus, the bacteriophage M13, which has been fluorescently labeled and anchored to a surface. It is demonstrated that the nanosensor can be calibrated and adapted for biological tissue, revealing WSS in micro-domains of cells that cannot be calculated accurately from bulk flow measurements. This method lends itself to a platform applicable to many applications in biology and microfluidics

Faithful chaperones

This review describes the properties of some rare eukaryotic chaperones that each assist in the folding of only one target protein. In particular, we describe (1) the tubulin cofactors, (2) p47, which assists in the folding of collagen, (3) α-hemoglobin stabilizing protein (AHSP), (4) the adenovirus L4-100 K protein, which is a chaperone of the major structural viral protein, hexon, and (5) HYPK, the huntingtin-interacting protein. These various-sized proteins (102–1,190 amino acids long) are all involved in the folding of oligomeric polypeptides but are otherwise functionally unique, as they each assist only one particular client. This raises a question regarding the biosynthetic cost of the high-level production of such chaperones. As the clients of faithful chaperones are all abundant proteins that are essential cellular or viral components, it is conceivable that this necessary metabolic expenditure withstood evolutionary pressure to minimize biosynthetic costs. Nevertheless, the complexity of the folding pathways in which these chaperones are involved results in error-prone processes. Several human disorders associated with these chaperones are discussed

Connector Inversion Probe Technology: A Powerful One-Primer Multiplex DNA Amplification System for Numerous Scientific Applications

We combined components of a previous assay referred to as Molecular Inversion Probe (MIP) with a complete gap filling strategy, creating a versatile powerful one-primer multiplex amplification system. As a proof-of-concept, this novel method, which employs a Connector Inversion Probe (CIPer), was tested as a genetic tool for pathogen diagnosis, typing, and antibiotic resistance screening with two distinct systems: i) a conserved sequence primer system for genotyping Human Papillomavirus (HPV), a cancer-associated viral agent and ii) screening for antibiotic resistance mutations in the bacterial pathogen Neisseria gonorrhoeae. We also discuss future applications and advances of the CIPer technology such as integration with digital amplification and next-generation sequencing methods. Furthermore, we introduce the concept of two-dimension informational barcodes, i.e. “multiplex multiplexing padlocks” (MMPs). For the readers' convenience, we also provide an on-line tutorial with user-interface software application CIP creator 1.0.1, for custom probe generation from virtually any new or established primer-pairs