Anti-oligomeric Abeta single-chain variable domain antibody blocks Abeta-induced toxicity against human neuroblastoma cells

The Amyloid-β (Aβ) peptide is a major component of the amyloid plaques associated with Alzheimer's disease (AD). Recent studies suggest that the most toxic forms of Aβ are small, soluble oligomeric aggregates. Here, we report the isolation and characterization of a single-chain variable domain (scFv) antibody isolated against oligomeric Aβ using a protocol developed in our laboratory that combines phage display technology and atomic force microscopy (AFM). Starting with a randomized, single framework phage display library, after three rounds of selection against oligomeric Aβ, we identified an scFv that bound oligomeric Aβ specifically, but not monomeric or fibrillar forms. The anti-oligomeric scFv inhibits Aβ aggregation and toxicity, and reduces the toxicity of preformed oligomeric Aβ towards human neuroblastoma cells. When used to probe samples of human brain tissue, the scFv reacted with AD tissue but not a healthy control or Parkinson's disease brain samples. The anti-oligomeric Aβ scFv therefore has potential therapeutic and diagnostic applications in specifically targeting or identifying the toxic morphologies of Aβ in AD brains.     

Sensitive genome-wide screen for low secondary enzymatic activities: the YjbQ family shows thiamin phosphate synthase activity

Contemporary enzymes are highly efficient and selective catalysts. However, due to the intrinsically very reactive nature of active sites, gratuitous secondary reactions are practically unavoidable. Consequently, even the smallest cell, with its limited enzymatic repertoire, has the potential to carry out numerous additional, very likely inefficient, secondary reactions. If selectively advantageous, secondary reactions could be the basis for the evolution of new fully functional enzymes. Here, we investigated if Escherichia coli has cryptic enzymatic activities related to thiamin biosynthesis. We selected this pathway because this vitamin is essential, but the cell's requirements are very small. Therefore, enzymes with very low activity could complement the auxotrophy of strains deleted of some bona fide thiamin biosynthetic genes. By overexpressing the E. coli's protein repertoire, we selected yjbQ, a gene that complemented a strain deleted of the thiamin phosphate synthase (TPS)-coding gene thiE. In vitro studies confirmed TPS activity, and by directed evolution experiments, this activity was enhanced. Structurally oriented mutagenesis allowed us to identify the putative active site. Remote orthologs of YjbQ from Thermotoga, Sulfolobus, and Pyrococcus were cloned and also showed thiamin auxotrophy complementation, indicating that the cryptic TPS activity is a property of this protein family. Interestingly, the thiE- and yjbQ-coded TPSs are analog enzymes with no structural similarity, reflecting distinct evolutionary origin. These results support the hypothesis that the enzymatic repertoire of a cell such as E. coli has the potential to perform vast amounts of alternative reactions, which could be exploited to evolve novel or more efficient catalysts.     

Substrate selectivity of the melibiose permease (MelY) from Enterobacter cloacae

We have examined the substrate selectivity of the melibiose permease (MelY) from Enterobacter cloacae in comparison with that of the lactose permease (LacY) from Escherichia coli. Both proteins catalyze active transport of lactose or melibiose with comparable affinity and capacity. However, MelY does not transport the analogue methyl-1-thio-β,d-galactopyranoside (TMG), which is a very efficient substrate in LacY. We show that MelY binds TMG and conserves Cys148 (helix V) as a TMG binding residue but fails to transport this ligand. Based on homology modeling, organization of the putative MelY sugar binding site is the same as that in LacY and residues irreplaceable for the symport mechanism are conserved. Moreover, only 15% of the residues where a single-Cys mutant is inactivated by site-directed alkylation differ in MelY. Using site-directed mutagenesis at these positions and engineered cross-homolog chimeras, we show that Val367, at the periplasmic end of transmembrane helix XI, contributes in defining the substrate selectivity profile. Replacement of Val367 with the MelY residue (Ala) leads to impairment of TMG uptake. Exchanging domains N6 and C6 between LacY and MelY also leads to impairment of TMG uptake. TMG uptake activity is restored by the re-introduction of a Val367 in the background of chimera N6(LacY)-C6(MelY). Much less prominent effects are found with the same mutants and chimeras for the transport of lactose or melibiose.     

Selective Cu2+ binding, redox silencing, and cytoprotective effects of the small heat shock proteins alphaA- and alphaB-crystallin

Oxidative stress and Cu²⁺ have been implicated in several neurodegenerative diseases and in cataract. Oxidative stress, as well as Cu²⁺, is also known to induce the expression of the small heat shock proteins α-crystallins. However, the role of α-crystallins in oxidative stress and in Cu²⁺-mediated processes is not clearly understood. We demonstrate using fluorescence and isothermal titration calorimetry that α-crystallins (αA- and αB-crystallin and its phosphorylation mimic, 3DαB-crystallin) bind Cu²⁺ with close to picomolar range affinity. The presence of other tested divalent cations such as Zn²⁺, Mg²⁺, and Ca²⁺ does not affect Cu²⁺ binding, indicating selectivity of the Cu²⁺-binding site(s) in α-crystallins. Cu²⁺ binding induces structural changes and increase in the hydrodynamic radii of α-crystallins. Cu²⁺ binding increases the stability of α-crystallins towards guanidinium chloride-induced unfolding. Chaperone activity of αA-crystallin increases significantly upon Cu²⁺ binding. α-Crystallins rescue amyloid beta peptide, Aβ_1-40, from Cu²⁺-induced aggregation in vitro. α-Crystallins inhibit Cu²⁺-induced oxidation of ascorbate and, hence, prevent the generation of reactive oxygen species. Interestingly, α-synuclein, a Cu²⁺-binding protein, does not inhibit this oxidation process significantly. We find that the Cu²⁺-sequestering (or redox-silencing) property of α-crystallins confers cytoprotection. To the best of our knowledge, this is the first study to reveal high affinity (close to picomolar) for Cu²⁺ binding and redox silencing of Cu²⁺ by any heat shock protein. Thus, our study ascribes a novel functional role to α-crystallins in Cu²⁺ homeostasis and helps in understanding their protective role in neurodegenerative diseases and cataract.     

A molecular dynamics study of the interaction of D-peptide amyloid inhibitors with their target sequence reveals a potential inhibitory pharmacophore conformation

The self-assembly of soluble proteins and peptides into β-sheet-rich oligomeric structures and insoluble fibrils is a hallmark of a large number of human diseases known as amyloid diseases. Drugs that are able to interfere with these processes may be able to prevent and/or cure these diseases. Experimental difficulties in the characterization of the intermediates involved in the amyloid formation process have seriously hampered the application of rational drug design approaches to the inhibition of amyloid formation and growth. Recently, short model peptide systems have proved useful in understanding the relationship between amino acid sequence and amyloid formation using both experimental and theoretical approaches. Moreover, short d-peptide sequences have been shown to specifically interfere with those short amyloid stretches in proteins, blocking oligomer formation or disassembling mature fibrils. With the aim of rationalizing which interactions drive the binding of inhibitors to nascent β-sheet oligomers, in this study, we have carried out extensive molecular dynamics simulations of the interaction of selected d-peptide sequences with oligomers of the target model sequence STVIIE. Structural analysis of the simulations helped to identify the molecular determinants of an inhibitory core whose conformational and physicochemical properties are actually shared by nonpeptidic small-molecule inhibitors of amyloidogenesis. Selection of one of these small molecules and experimental validation against our model system proved that it was indeed an effective inhibitor of fibril formation by the STVIIE sequence, supporting theoretical predictions. We propose that the inhibitory determinants derived from this work be used as structural templates in the development of pharmacophore models for the identification of novel nonpeptidic inhibitors of aggregation.     

Rapid incorporation of functional rhodopsin into nanoscale apolipoprotein bound bilayer (NABB) particles

Human apolipoprotein A-I (apo A-I) and its engineered constructs form discoidal lipid bilayers upon interaction with lipids in vitro. We now report the cloning, expression, and purification of apo A-I derived from zebrafish (Danio rerio), which combines with phospholipids to form similar discoidal bilayers and may prove to be superior to human apo A-I constructs for rapid reconstitution of seven-transmembrane helix receptors into nanoscale apolipoprotein bound bilayers (NABBs). We characterized NABBs by gel-filtration chromatography, native polyacrylamide gradient gel electrophoresis, UV-visible photobleaching difference spectroscopy, and fluorescence spectroscopy. We used electron microscopy to determine the stoichiometry and orientation of rhodopsin (rho)-containing NABBs prepared under various conditions and correlated stability and signaling efficiency of rho in NABBs with either one or two receptors. We discovered that the specific activity of G protein coupling for single rhos sequestered in individual NABBs was nearly identical with that of two rhos per NABB under conditions where stoichiometry and orientation could be inferred by electron microscopy imaging. Thermal stability of rho in NABBs was superior to that of rho in various commonly used detergents. We conclude that the NABB system using engineered zebrafish apo A-I is a native-like membrane mimetic system for G-protein-coupled receptors and discuss strategies for rapid incorporation of expressed membrane proteins into NABBs.     

Mutations enhance the aggregation propensity of the Alzheimer's A beta peptide

Aggregation of the amyloid β (Aβ) peptide plays a key role in the molecular etiology of Alzheimer’s disease. Despite the importance of this process, the relationship between the sequence of Aβ and the propensity of the peptide to aggregate has not been fully elucidated. The sequence determinants of aggregation can be revealed by probing the ability of amino acid substitutions (mutations) to increase or decrease aggregation. Numerous mutations that decrease aggregation have been isolated by laboratory-based studies. In contrast, very few mutations that increase aggregation have been reported, and most of these were isolated from rare individuals with early-onset familial Alzheimer’s disease. To augment the limited data set of clinically derived mutations, we developed an artificial genetic screen to isolate novel mutations that increase aggregation propensity. The screen relies on the expression of Aβ-green fluorescent protein fusion in Escherichia coli. In this fusion, the ability of the green fluorescent protein reporter to fold and fluoresce is inversely correlated with the aggregation propensity of the Aβ sequence. Implementation of this screen enabled the isolation of 20 mutant versions of Aβ with amino acid substitutions at 17 positions in the 42-residue sequence of Aβ. Biophysical studies of synthetic peptides corresponding to sequences isolated by the screen confirm the increased aggregation propensity and amyloidogenic behavior of the mutants. The mutations were isolated using an unbiased screen that makes no assumptions about the sequence determinants of aggregation. Nonetheless, all 16 of the most aggregating mutants contain substitutions that reduce charge and/or increase hydrophobicity. These findings provide compelling evidence supporting the hypothesis that sequence hydrophobicity is a major determinant of Aβ aggregation.     

Compensatory and long-range changes in picosecond-nanosecond main-chain dynamics upon complex formation: 15N relaxation analysis of the free and bound states of the ubiquitin-like domain of human plexin-B1 and the small GTPase Rac1

The formation of a complex between Rac1 and the cytoplasmic domain of plexin-B1 is one of the first documented cases of a direct interaction between a small guanosine 5′-triphosphatase (GTPase) and a transmembrane receptor. Structural studies have begun to elucidate the role of this interaction for the signal transduction mechanism of plexins. Mapping of the Rac1 GTPase surface that contacts the Rho GTPase binding domain of plexin-B1 by solution NMR spectroscopy confirms the plexin domain as a GTPase effector protein. Regions neighboring the GTPase switch I and II regions are also involved in the interaction and there is considerable interest to examine the changes in protein dynamics that take place upon complex formation. Here we present main-chain nitrogen-15 relaxation measurements for the unbound proteins as well as for the Rho GTPase binding domain and Rac1 proteins in their complexed state. Derived order parameters, S², show that considerable motions are maintained in the bound state of plexin. In fact, some of the changes in S² on binding appear compensatory, exhibiting decreased as well as increased dynamics. Fluctuations in Rac1, already a largely rigid protein on the picosecond-nanosecond timescale, are overall diminished, but isomerization dynamics in the switch I and II regions of the GTPase are retained in the complex and appear to be propagated to the bound plexin domain. Remarkably, fluctuations in the GTPase are attenuated at sites, including helices α6 (the Rho-specific insert helix), α7 and α8, that are spatially distant from the interaction region with plexin. This effect of binding on long-range dynamics appears to be communicated by hinge sites and by subtle conformational changes in the protein. Similar to recent studies on other systems, we suggest that dynamical protein features are affected by allosteric mechanisms. Altered protein fluctuations are likely to prime the Rho GTPase-plexin complex for interactions with additional binding partners.     

蜂胶黄酮对大鼠脑缺血再灌注损伤后IL-1β、IL-6和TNF-α含量的影响

目的探讨蜂胶黄酮对脑缺血再灌注损伤的保护作用。方法采用大鼠大脑中动脉栓塞模型（MCAO）,研究蜂胶黄酮对脑缺血再灌注脑梗死体积和行为学评分的影响,对脑组织内IL-1β、IL-6和TNF-α含量的影响。结果蜂胶黄酮能够减少MCAO脑梗死体积和行为学评分,降低脑组织中IL-1β、IL-6和TNF-α的含量（P〈0．05）。结论蜂胶黄酮对大鼠脑缺血再灌注损伤有一定保护作用,其作用机制与降低脑组织中IL-1β、IL-6和TNF-α含量有关。     

PPARβ在中枢神经系统中的作用研究进展

PPARβ是配体活化的核转录因子,属核受体超家族成员。PPARβ在哺乳动物体内表达十分丰富,日前对PPARβ的研究比较少,但现有的研究表明PPARβ可能参与了机体多种生理和病理过程。本文将对PPARβ的生物学特征及其在中枢神经系统中的意义作一综述。