Application of a benchtop colorimetric method for quantification of blood propofol levels.

Quantification of plasma propofol (2,6-diisopropylphenol) in the context of clinical anaesthesia is challenging because of the need for offline blood sample processing using specialised laboratory equipment and techniques. In this study we sought to refine a simple procedure using solid phase extraction and colorimetric analysis into a benchtop protocol for accurate blood propofol measurement. The colorimetric method based on the reaction of phenols (e.g. propofol) with Gibbs reagent was first tested in 10% methanol samples (n = 50) containing 0.5-6.0 µg/mL propofol. Subsequently, whole blood samples (n = 15) were spiked to known propofol concentrations and processed using reverse phase solid phase extraction (SPE) and colorimetric analysis. The standard deviation of the difference between known and measured propofol concentrations in the methanol samples was 0.11 µg/mL, with limits of agreement of - 0.21 to 0.22 µg/mL. For the blood-processed samples, the standard deviation of the difference between known and measured propofol concentrations was 0.09 µg/mL, with limits of agreement - 0.18 to 0.17 µg/mL. Quantification of plasma propofol with an error of less than 0.2 µg/mL is achievable with a simple and inexpensive benchtop method.

Surface-enhanced Raman scattering sensing platform for detecting amyloid-ß peptide interaction with an aggregation inhibitor.

Soluble, small amyloid-ß oligomers (AßO) are recognized as significant contributors to the pathology of Alzheimer's disease (AD). Although drugs for treating AD symptoms have been approved, no therapy targeting amyloid-ß (Aß) capable of modifying the course of the disease is available. In an effort to develop a label-free method for screening new anti-AD therapeutic agents, we show the use of a surface-enhanced Raman scattering (SERS) active substrate for detecting the interactions between Aß peptides and spin-labeled fluorine (SLF), a peptide aggregation inhibitor. Changes in the peak positions and intensity ratios of two spectral peaks near 1600cm-1 and 2900cm-1 can be used to monitor the molecular interactions between SLF and Aß. This study demonstrates the potential of SERS spectroscopy for rapidly screening and identifying new anti-Aß therapeutic agents.

A Bifunctional Anti-Amyloid Blocks Oxidative Stress and the Accumulation of Intraneuronal Amyloid-Beta.

There is growing recognition regarding the role of intracellular amyloid beta (Aß) in the Alzheimer's disease process, which has been linked with aberrant signaling and the disruption of protein degradation mechanisms. Most notably, intraneuronal Aß likely underlies the oxidative stress and mitochondrial dysfunction that have been identified as key elements of disease progression. In this study, we employed fluorescence imaging to explore the ability of a bifunctional small molecule to reduce aggregates of intracellular Aß and attenuate oxidative stress. Structurally, this small molecule is comprised of a nitroxide spin label linked to an amyloidophilic fluorene and is known as spin-labeled fluorene (SLF). The effect of the SLF on intracellular Aß accumulation and oxidative stress was measured in MC65 cells, a human neuronal cell line with inducible expression of the amyloid precursor protein and in the N2a neuronal cell line treated with exogenous Aß. Super-resolution microscopy imaging showed SLF decreases the accumulation of intracellular Aß. Confocal microscopy imaging of MC65 cells treated with a reactive oxygen species (ROS)-sensitive dye demonstrated SLF significantly reduces the intracellular Aß-induced ROS signal. In order to determine the contributions of the separate SLF moieties to these protective activities, experiments were also carried out on cells with nitroxides lacking the Aß targeting domain or fluorene derivatives lacking the nitroxide functionality. The findings support a synergistic effect of SLF in counteracting both the conformational toxicity of both endogenous and exogenous Aß, its promotion of ROS, and Aß metabolism. Furthermore, these studies demonstrate an intimate link between ROS production and Aß oligomer formation.

Lipotoxic brain microvascular injury is mediated by activating transcription factor 3-dependent inflammatory and oxidative stress pathways.

Dysfunction of the cerebrovasculature plays an important role in vascular cognitive impairment (VCI). Lipotoxic injury of the systemic endothelium in response to hydrolyzed triglyceride-rich lipoproteins (TGRLs; TGRL lipolysis products) or a high-fat Western diet (WD) suggests similar mechanisms may be present in brain microvascular endothelium. We investigated the hypothesis that TGRL lipolysis products cause lipotoxic injury to brain microvascular endothelium by generating increased mitochondrial superoxide radical generation, upregulation of activating transcription factor 3 (ATF3)-dependent inflammatory pathways, and activation of cellular oxidative stress and apoptotic pathways. Human brain microvascular endothelial cells were treated with human TGRL lipolysis products that induced intracellular lipid droplet formation, mitochondrial superoxide generation, ATF3-dependent transcription of proinflammatory, stress response, and oxidative stress genes, as well as activation of proapoptotic cascades. Male apoE knockout mice were fed a high-fat/high-cholesterol WD for 2 months, and brain microvessels were isolated by laser capture microdissection. ATF3 gene transcription was elevated 8-fold in the hippocampus and cerebellar brain region of the WD-fed animals compared with chow-fed control animals. The microvascular injury phenotypes observed in vitro and in vivo were similar. ATF3 plays an important role in mediating brain microvascular responses to acute and chronic lipotoxic injury and may be an important preventative and therapeutic target for endothelial dysfunction in VCI.

Protective spin-labeled fluorenes maintain amyloid beta peptide in small oligomers and limit transitions in secondary structure.

Alzheimer's disease is characterized by the presence of extracellular plaques comprised of amyloid beta (Aß) peptides. Soluble oligomers of the Aß peptide underlie a cascade of neuronal loss and dysfunction associated with Alzheimer's disease. Single particle analyses of Aß oligomers in solution by fluorescence correlation spectroscopy (FCS) were used to provide real-time descriptions of how spin-labeled fluorenes (SLFs; bi-functional small molecules that block the toxicity of Aß) prevent and disrupt oligomeric assemblies of Aß in solution. Furthermore, the circular dichroism (CD) spectrum of untreated Aß shows a continuous, progressive change over a 24-hour period, while the spectrum of Aß treated with SLF remains relatively constant following initial incubation. These findings suggest the conformation of Aß within the oligomer provides a complementary determinant of Aß toxicity in addition to oligomer growth and size. Although SLF does not produce a dominant state of secondary structure in Aß, it does induce a net reduction in beta secondary content compared to untreated samples of Aß. The FCS results, combined with electron paramagnetic resonance spectroscopy and CD spectroscopy, demonstrate SLFs can inhibit the growth of Aß oligomers and disrupt existing oligomers, while retaining Aß as a population of smaller, yet largely disordered oligomers.

Molecular crowding impacts the structure of apolipoprotein A-I with potential implications on in vivo metabolism and function.

The effect molecular crowding, defined as the volume exclusion exerted by one soluble inert molecule upon another soluble molecule, has on the structure and self-interaction of lipid-free apoA-I were explored. The influence of molecular crowding on lipid-free apoA-I oligomerization and internal dynamics has been analyzed using electron paramagnetic resonance (EPR) spectroscopy measurements of nitroxide spin label at selected positions throughout the protein sequence and at varying concentrations of the crowding agent Ficoll-70. The targeted positions include sites previously shown to be sensitive for detecting intermolecular interaction via spin-spin coupling. Circular dichroism was used to study secondary structural changes in lipid-free apoA-I imposed by increasing concentrations of the crowding agent. Crosslinking and SDS-PAGE gel analysis was employed to further characterize the role molecular crowding plays in inducing apoA-I oligomerization. It was concluded that the dynamic apoA-I structure and oligomeric state was altered in the presence of the crowding agent. It was also found that the C-terminal was slightly more sensitive to molecular crowding. Finally, the data described the region around residue 217 in the C-terminal domain of apoA-I as the most sensitive reporter of the crowding-induced self-association of apoA-I. The implications of this behavior to in vivo functionality are discussed. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 683-692, 2016.

Antimitochondrial antibody recognition and structural integrity of the inner lipoyl domain of the E2 subunit of pyruvate dehydrogenase complex.

Antimitochondrial autoantibodies (AMAs), the serological hallmark of primary biliary cirrhosis, are directed against the lipoyl domain of the E2 subunit of pyruvate dehydrogenase (PDC-E2). However, comprehensive analysis of the amino acid residues of PDC-E2 lipoyl ß-sheet with AMA specificity is lacking. In this study, we postulated that specific residues within the lipoyl domain are critical to AMA recognition by maintaining conformational integrity. We systematically replaced each of 19 residue peptides of the inner lipoyl domain with alanine and analyzed these mutants for reactivities against 60 primary biliary cirrhosis and 103 control sera. Based on these data, we then constructed mutants with two, three, or four replacements and, in addition, probed the structure of the substituted domains using thiol-specific spin labeling and electron paramagnetic resonance (EPR) of a (5)Ile→Ala and (12)Ile→Ala double mutant. Single alanine replacement at (5)Ile, (12)Ile, and (15)Glu significantly reduced AMA recognition. In addition, mutants with two, three, or four replacements at (5)Ile, (12)Ile, and (15)Glu reduced AMA reactivity even further. Indeed, EPR reveals a highly flexible structure within the (5)Ile and (12)Ile double-alanine mutant. Autoreactivity is largely focused on specific residues in the PDC-E2 lipoyl domain critical in maintaining the lipoyl loop conformation necessary for AMA recognition. Collectively, the AMA binding studies and EPR analysis demonstrate the necessity of the lipoyl ß-sheet structural conformation in anti-PDC-E2 recognition.

Binding of apolipoprotein E inhibits the oligomer growth of amyloid-ß peptide in solution as determined by fluorescence cross-correlation spectroscopy.

One of the primary neuropathological hallmarks of Alzheimer disease is the presence of extracellular amyloid plaques resulting from the aggregation of amyloid-ß (Aß) peptides. The intrinsic disorder of the Aß peptide drives self-association and progressive reordering of the conformation in solution, and this dynamic distribution of Aß complicates biophysical studies. This property poses a challenge for understanding the interaction of Aß with apolipoprotein E (apoE). ApoE plays a pivotal role in the aggregation and clearance of Aß peptides in the brain, and the ε4 allele of APOE is the most significant known genetic modulator of Alzheimer risk. Understanding the interaction between apoE and Aß will provide insight into the mechanism by which different apoE isoforms determine Alzheimer disease risk. Here we applied alternating laser excitation fluorescence cross-correlation spectroscopy to observe the single molecule interaction of Aß with apoE in the hydrated state. The diffusion time of freely diffusing Aß in the absence of apoE shows significant self-aggregation, whereas in the presence of apoE, binding of the protein results in a more stable complex. These results show that apoE slows down the oligomerization of Aß in solution and provide direct insight into the process by which apoE influences the deposition and clearance of Aß peptides in the brain. Furthermore, by developing an approach to remove signals arising from very large Aß aggregates, we show that real-time single particle observations provide access to information regarding the fraction of apoE bound and the stoichiometry of apoE and Aß in the complex.

Molecular dynamics simulation of dipalmitoylphosphatidylcholine modified with a MTSL nitroxide spin label in a lipid membrane.

We investigate the interaction between dipalmitoylphosphatidylcholine (DPPC) and a nitroxide spin label in order to understand its influences on lipid structure and dynamics using molecular dynamics simulations. The system was modified by covalently attaching nitroxide spin labels to the headgroups of two DPPC molecules. (S-(2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl)methyl methanesulfonothioate) (MTSL) was used as the spin label. The label position and dynamics were analyzed as was the impact of the modified DPPC on the structure of the surrounding lipids. The modified DPPC molecules locate closer to the center of the membrane than unmodified DPPC molecules. The rotation of the spin label is unrestricted, but there are favored orientations. MTSL depresses the deuterium order parameters of the carbon atoms close to the headgroup in surrounding DPPC molecules. The spin label has no impact on order parameters of carbon atoms at the end of the lipid tails. The lateral diffusion constant of the modified DPPC is indistinguishable from unmodified DPPC molecules. These novel computational results suggest an experimental validation.

Analysis of lipid phase behavior and protein conformational changes in nanolipoprotein particles upon entrapment in sol-gel-derived silica.

The entrapment of nanolipoprotein particles (NLPs) and liposomes in transparent, nanoporous silica gel derived from the precursor tetramethylorthosilicate was investigated. NLPs are discoidal patches of lipid bilayer that are belted by amphiphilic scaffold proteins and have an average thickness of 5 nm. The NLPs in this work had a diameter of roughly 15 nm and utilized membrane scaffold protein (MSP), a genetically altered variant of apolipoprotein A-I. Liposomes have previously been examined inside of silica sol-gels and have been shown to exhibit instability. This is attributed to their size (∼150 nm) and altered structure and constrained lipid dynamics upon entrapment within the nanometer-scale pores (5-50 nm) of the silica gel. By contrast, the dimensional match of NLPs with the intrinsic pore sizes of silica gel opens the possibility for their entrapment without disruption. Here we demonstrate that NLPs are more compatible with the nanometer-scale size of the porous environment by analysis of lipid phase behavior via fluorescence anisotropy and analysis of scaffold protein secondary structure via circular dichroism spectroscopy. Our results showed that the lipid phase behavior of NLPs entrapped inside of silica gel display closer resemblance to its solution behavior, more so than liposomes, and that the MSP in the NLPs maintain the high degree of α-helix secondary structure associated with functional protein-lipid interactions after entrapment. We also examined the effects of residual methanol on lipid phase behavior and the size of NLPs and found that it exerts different influences in solution and in silica gel; unlike in free solution, silica entrapment may be inhibiting NLP size increase and/or aggregation. These findings set precedence for a bioinorganic hybrid nanomaterial that could incorporate functional integral membrane proteins.

Conservation of apolipoprotein A-I's central domain structural elements upon lipid association on different high-density lipoprotein subclasses.

The antiatherogenic properties of apolipoprotein A-I (apoA-I) are derived, in part, from lipidation-state-dependent structural elements that manifest at different stages of apoA-I's progression from lipid-free protein to spherical high-density lipoprotein (HDL). Previously, we reported the structure of apoA-I's N-terminus on reconstituted HDLs (rHDLs) of different sizes. We have now investigated at the single-residue level the conformational adaptations of three regions in the central domain of apoA-I (residues 119-124, 139-144, and 164-170) upon apoA-I lipid binding and HDL formation. An important function associated with these residues of apoA-I is the activation of lecithin:cholesterol acyltransferase (LCAT), the enzyme responsible for catalyzing HDL maturation. Structural examination was performed by site-directed tryptophan fluorescence and spin-label electron paramagnetic resonance spectroscopies for both the lipid-free protein and rHDL particles 7.8, 8.4, and 9.6 nm in diameter. The two methods provide complementary information about residue side chain mobility and molecular accessibility, as well as the polarity of the local environment at the targeted positions. The modulation of these biophysical parameters yielded new insight into the importance of structural elements in the central domain of apoA-I. In particular, we determined that the loosely lipid-associated structure of residues 134-145 is conserved in all rHDL particles. Truncation of this region completely abolished LCAT activation but did not significantly affect rHDL size, reaffirming the important role of this structural element in HDL function.

Double electron-electron resonance probes Ca²âº-induced conformational changes and dimerization of recoverin.

Recoverin, a member of the neuronal calcium sensor (NCS) branch of the calmodulin superfamily, is expressed in retinal photoreceptor cells and serves as a calcium sensor in vision. Ca²âº-induced conformational changes in recoverin cause extrusion of its covalently attached myristate (termed Ca²âº-myristoyl switch) that promotes translocation of recoverin to disk membranes during phototransduction in retinal rod cells. Here we report double electron-electron resonance (DEER) experiments on recoverin that probe Ca²âº-induced changes in distance as measured by the dipolar coupling between spin-labels strategically positioned at engineered cysteine residues on the protein surface. The DEER distance between nitroxide spin-labels attached at C39 and N120C is 2.5 ± 0.1 nm for Ca²âº-free recoverin and 3.7 ± 0.1 nm for Ca²âº-bound recoverin. An additional DEER distance (5-6 nm) observed for Ca²âº-bound recoverin may represent an intermolecular distance between C39 and N120. ¹5N NMR relaxation analysis and CW-EPR experiments both confirm that Ca²âº-bound recoverin forms a dimer at protein concentrations above 100 µM, whereas Ca²âº-free recoverin is monomeric. We propose that Ca²âº-induced dimerization of recoverin at the disk membrane surface may play a role in regulating Ca²âº-dependent phosphorylation of dimeric rhodopsin. The DEER approach will be useful for elucidating dimeric structures of NCS proteins in general for which Ca²âº-induced dimerization is functionally important but not well understood.

The structure of vimentin linker 1 and rod 1B domains characterized by site-directed spin-labeling electron paramagnetic resonance (SDSL-EPR) and X-ray crystallography.

Despite the passage of ∼30 years since the complete primary sequence of the intermediate filament (IF) protein vimentin was reported, the structure remains unknown for both an individual protomer and the assembled filament. In this report, we present data describing the structure of vimentin linker 1 (L1) and rod 1B. Electron paramagnetic resonance spectra collected from samples bearing site-directed spin labels demonstrate that L1 is not a flexible segment between coiled-coils (CCs) but instead forms a rigid, tightly packed structure. An x-ray crystal structure of a construct containing L1 and rod 1B shows that it forms a tetramer comprising two equivalent parallel CC dimers that interact with one another in the form of a symmetrical anti-parallel dimer. Remarkably, the parallel CC dimers are themselves asymmetrical, which enables them to tetramerize rather than undergoing higher order oligomerization. This functionally vital asymmetry in the CC structure, encoded in the primary sequence of rod 1B, provides a striking example of evolutionary exploitation of the structural plasticity of proteins. EPR and crystallographic data consistently suggest that a very short region within L1 represents a minor local distortion in what is likely to be a continuous CC from the end of rod 1A through the entirety of rod 1B. The concordance of this structural model with previously published cross-linking and spectral data supports the conclusion that the crystallographic oligomer represents a native biological structure.

Small molecule structure correctors abolish detrimental effects of apolipoprotein E4 in cultured neurons.

Apolipoprotein E4 (apoE4), the major genetic risk factor for late onset Alzheimer disease, assumes a pathological conformation, intramolecular domain interaction. ApoE4 domain interaction mediates the detrimental effects of apoE4, including decreased mitochondrial cytochrome c oxidase subunit 1 levels, reduced mitochondrial motility, and reduced neurite outgrowth in vitro. Mutant apoE4 (apoE4-R61T) lacks domain interaction, behaves like apoE3, and does not cause detrimental effects. To identify small molecules that inhibit domain interaction (i.e. structure correctors) and reverse the apoE4 detrimental effects, we established a high throughput cell-based FRET primary assay that determines apoE4 domain interaction and secondary cell- and function-based assays. Screening a ChemBridge library with the FRET assay identified CB9032258 (a phthalazinone derivative), which inhibits domain interaction in neuronal cells. In secondary functional assays, CB9032258 restored mitochondrial cytochrome c oxidase subunit 1 levels and rescued impairments of mitochondrial motility and neurite outgrowth in apoE4-expressing neuronal cells. These benefits were apoE4-specific and dose-dependent. Modifying CB9032258 yielded well defined structure-activity relationships and more active compounds with enhanced potencies in the FRET assay (IC(50) of 23 and 116 nm, respectively). These compounds efficiently restored functional activities of apoE4-expressing cells in secondary assays. An EPR binding assay showed that the apoE4 structure correction resulted from direct interaction of a phthalazinone. With these data, a six-feature pharmacophore model was constructed for future drug design. Our results serve as a proof of concept that pharmacological intervention with apoE4 structure correctors negates apoE4 detrimental effects in neuronal cells and could be further developed as an Alzheimer disease therapeutic.

The "beta-clasp" model of apolipoprotein A-I--a lipid-free solution structure determined by electron paramagnetic resonance spectroscopy.

Apolipoprotein A-I (apoA-I) is the major protein component of high density lipoproteins (HDL) and plays a central role in cholesterol metabolism. The lipid-free/lipid-poor form of apoA-I is the preferred substrate for the ATP-binding cassette transporter A1 (ABCA1). The interaction of apoA-I with ABCA1 leads to the formation of cholesterol laden high density lipoprotein (HDL) particles, a key step in reverse cholesterol transport and the maintenance of cholesterol homeostasis. Knowledge of the structure of lipid-free apoA-I is essential to understanding its critical interaction with ABCA1 and the molecular mechanisms underlying HDL biogenesis. We therefore examined the structure of lipid-free apoA-I by electron paramagnetic resonance spectroscopy (EPR). Through site directed spin label EPR, we mapped the secondary structure of apoA-I and identified sites of spin coupling as residues 26, 44, 64, 167, 217 and 226. We capitalize on the fact that lipid-free apoA-I self-associates in an anti-parallel manner in solution. We employed these sites of spin coupling to define the central plane in the dimeric apoA-I complex. Applying both the constraints of dipolar coupling with the EPR-derived pattern of solvent accessibility, we assembled the secondary structure into a tertiary context, providing a solution structure for lipid-free apoA-I. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).

Characterization of the biochemical and biophysical properties of the Saccharomyces cerevisiae phosphate transporter Pho89.

In Saccharomyces cerevisiae, Pho89 mediates a cation-dependent transport of Pi across the plasma membrane. This integral membrane protein belongs to the Inorganic Phosphate Transporter (PiT) family, a group that includes the mammalian Na(+)/Pi cotransporters Pit1 and Pit2. Here we report that the Pichia pastoris expressed recombinant Pho89 was purified in the presence of Foscholine-12 and functionally reconstituted into proteoliposomes with a similar substrate specificity as observed in an intact cell system. The alpha-helical content of the Pho89 protein was estimated to 44%. EPR analysis showed that purified Pho89 protein undergoes conformational change upon addition of substrate.

Structure of apolipoprotein A-I N terminus on nascent high density lipoproteins.

Apolipoprotein A-I (apoA-I) is the major protein component of high density lipoproteins (HDL) and a critical element of cholesterol metabolism. To better elucidate the role of the apoA-I structure-function in cholesterol metabolism, the conformation of the apoA-I N terminus (residues 6-98) on nascent HDL was examined by electron paramagnetic resonance (EPR) spectroscopic analysis. A series of 93 apoA-I variants bearing single nitroxide spin label at positions 6-98 was reconstituted onto 9.6-nm HDL particles (rHDL). These particles were subjected to EPR spectral analysis, measuring regional flexibility and side chain solvent accessibility. Secondary structure was elucidated from side-chain mobility and molecular accessibility, wherein two major α-helical domains were localized to residues 6-34 and 50-98. We identified an unstructured segment (residues 35-39) and a ß-strand (residues 40-49) between the two helices. Residues 14, 19, 34, 37, 41, and 58 were examined by EPR on 7.8, 8.4, and 9.6 nm rHDL to assess the effect of particle size on the N-terminal structure. Residues 14, 19, and 58 showed no significant rHDL size-dependent spectral or accessibility differences, whereas residues 34, 37, and 41 displayed moderate spectral changes along with substantial rHDL size-dependent differences in molecular accessibility. We have elucidated the secondary structure of the N-terminal domain of apoA-I on 9.6 nm rHDL (residues 6-98) and identified residues in this region that are affected by particle size. We conclude that the inter-helical segment (residues 35-49) plays a role in the adaptation of apoA-I to the particle size of HDL.

Structural modeling and electron paramagnetic resonance spectroscopy of the human Na+/H+ exchanger isoform 1, NHE1.

We previously presented evidence that transmembrane domain (TM) IV and TM X-XI are important for inhibitor binding and ion transport by the human Na(+)/H(+) exchanger, hNHE1 (Pedersen, S. F., King, S. A., Nygaard, E. B., Rigor, R. R., and Cala, P. M. (2007) J. Biol. Chem. 282, 19716-19727). Here, we present a structural model of the transmembrane part of hNHE1 that further supports this conclusion. The hNHE1 model was based on the crystal structure of the Escherichia coli Na(+)/H(+) antiporter, NhaA, and previous cysteine scanning accessibility studies of hNHE1 and was validated by EPR spectroscopy of spin labels in TM IV and TM XI, as well as by functional analysis of hNHE1 mutants. Removal of all endogenous cysteines in hNHE1, introduction of the mutations A173C (TM IV) and/or I461C (TM XI), and expression of the constructs in mammalian cells resulted in functional hNHE1 proteins. The distance between these spin labels was ∼15 A, confirming that TM IV and TM XI are in close proximity. This distance was decreased both at pH 5.1 and in the presence of the NHE1 inhibitor cariporide. A similar TM IV·TM XI distance and a similar change upon a pH shift were found for the cariporide-insensitive Pleuronectes americanus (pa) NHE1; however, in paNHE1, cariporide had no effect on TM IV·TM XI distance. The central role of the TM IV·TM XI arrangement was confirmed by the partial loss of function upon mutation of Arg(425), which the model predicts stabilizes this arrangement. The data are consistent with a role for TM IV and TM XI rearrangements coincident with ion translocation and inhibitor binding by hNHE1.

Proteinuria decreases tissue lipoprotein receptor levels resulting in altered lipoprotein structure and increasing lipid levels.

Rats with nephrotic syndrome (NS) have a fivefold increase in lipids and a similar decrease in triglyceride-rich lipoprotein (TRL) clearance. Lipoprotein lipase (LPL) is reduced both in NS and in the Nagase analbuminemic rat. These rats have nearly normal triglyceride levels and TRL clearance, suggesting that reduction in LPL alone is insufficient to cause increased TRL levels. Apolipoprotein E (apoE) was decreased in lipoprotein fractions in NS, but not in analbuminemia. Here we tested whether decreased apoE binding to lipoproteins in NS contributes to hyperlipidemia by decreasing their affinity for lipoprotein receptors. Plasma apoE was increased 60% in both NS and analbuminemia compared with control (CTRL) as a result of a 60% decreased apoE clearance. Very-low-density lipoprotein and high-density lipoprotein in NS had significantly less apoE per mole of phospholipid compared with analbuminemia or CTRL and significantly greater lipid content; however, apoE binding did not differ by lipoprotein class or group. There was a significant reduction of receptors for lipoproteins in nearly all tissues in NS compared with CTRL and analbuminemia. Thus, apoE within lipoprotein fractions was reduced by dilution resulting from expansion of the lipid fraction due to decreased lipolysis and not to differing affinity for apoE. Decreased lipoprotein receptors result from proteinuria and contribute to hyperlipidemia in NS.

Novel Stilbene-Nitroxyl Hybrid Compounds Display Discrete Modulation of Amyloid Beta Toxicity and Structure.

Several neurodegenerative diseases are driven by misfolded proteins that assemble into soluble aggregates. These "toxic oligomers" have been associated with a plethora of cellular dysfunction and dysregulation, however the structural features underlying their toxicity are poorly understood. A major impediment to answering this question relates to the heterogeneous nature of the oligomers, both in terms of structural disorder and oligomer size. This not only complicates elucidating the molecular etiology of these disorders, but also the druggability of these targets as well. We have synthesized a class of bifunctional stilbenes to modulate both the conformational toxicity within amyloid beta oligomers (AßO) and the oxidative stress elicited by AßO. Using a neuronal culture model, we demonstrate this bifunctional approach has the potential to counter the molecular pathogenesis of Alzheimer's disease in a powerful, synergistic manner. Examination of AßO structure by various biophysical tools shows that each stilbene candidate uniquely alters AßO conformation and toxicity, providing insight towards the future development of structural correctors for AßO. Correlations of AßO structural modulation and bioactivity displayed by each provides insights for future testing in vivo. The multi-target activity of these hybrid molecules represents a highly advantageous feature for disease modification in Alzheimer's, which displays a complex, multifactorial etiology. Importantly, these novel small molecules intervene with intraneuronal AßO, a necessary feature to counter the cycle of dysregulation, oxidative stress and inflammation triggered during the earliest stages of disease progression.