Polar/Ionizable Residues in Transmembrane Segments: Effects on Helix-Helix Packing

The vast majority of membrane proteins are anchored to biological membranes through hydrophobic α-helices. Sequence analysis of high-resolution membrane protein structures show that ionizable amino acid residues are present in transmembrane (TM) helices, often with a functional and/or structural role. Here, using as scaffold the hydrophobic TM domain of the model membrane protein glycophorin A (GpA), we address the consequences of replacing specific residues by ionizable amino acids on TM helix insertion and packing, both in detergent micelles and in biological membranes. Our findings demonstrate that ionizable residues are stably inserted in hydrophobic environments, and tolerated in the dimerization process when oriented toward the lipid face, emphasizing the complexity of protein-lipid interactions in biological membranes

Inhibition of Amyloid-β Aggregation and Caspase-3 Activation by the \u3ci\u3eGinkgo biloba\u3c/i\u3e Extract EGb761

Standardized extract from the leaves of the Ginkgo biloba tree, labeled EGb761, has been used in clinical trials for its beneficial effects on brain functions, particularly in connection with age-related dementias and Alzheimer\u27s disease (AD). Substantial experimental evidence indicates that EGb761 protects against neuronal damage from a variety of insults, but its cellular and molecular mechanisms remain unknown. Using a neuroblastoma cell line stably expressing an AD-associated double mutation, we report that EGb761 inhibits formation of amyloid-β (Aβ) fibrils, which are the diagnostic, and possibly causative, feature of AD. The decreased Aβ fibrillogenesis in the presence of EGb761 was observed both in the conditioned medium of this Aβ-secreting cell line and in solution in vitro. In the cells, EGb761 significantly attenuated mitochondrion-initiated apoptosis and decreased the activity of caspase 3, a key enzyme in the apoptosis cell-signaling cascade. These results suggest that (i) neuronal damage in AD might be due to two factors: a direct Aβ toxicity and the apoptosis initiated by the mitochondria; and (ii) multiple cellular and molecular neuroprotective mechanisms, including attenuation of apoptosis and direct inhibition of Aβ aggregation, underlie the neuroprotective effects of EGb761

Characterisation of Peptide Microarrays for Studying Antibody-Antigen Binding Using Surface Plasmon Resonance Imagery

BACKGROUND: Non-specific binding to biosensor surfaces is a major obstacle to quantitative analysis of selective retention of analytes at immobilized target molecules. Although a range of chemical antifouling monolayers has been developed to address this problem, many macromolecular interactions still remain refractory to analysis due to the prevalent high degree of non-specific binding. We describe how we use the dynamic process of the formation of self assembling monolayers and optimise physical and chemical properties thus reducing considerably non-specific binding and allowing analysis of specific binding of analytes to immobilized target molecules. METHODOLOGY/PRINCIPAL FINDINGS: We illustrate this approach by the production of specific protein arrays for the analysis of interactions between the 65kDa isoform of human glutamate decarboxylase (GAD65) and a human monoclonal antibody. Our data illustrate that we have effectively eliminated non-specific interactions with the surface containing the immobilised GAD65 molecules. The findings have several implications. First, this approach obviates the dubious process of background subtraction and gives access to more accurate kinetic and equilibrium values that are no longer contaminated by multiphase non-specific binding. Second, an enhanced signal to noise ratio increases not only the sensitivity but also confidence in the use of SPR to generate kinetic constants that may then be inserted into van't Hoff type analyses to provide comparative DeltaG, DeltaS and DeltaH values, making this an efficient, rapid and competitive alternative to ITC measurements used in drug and macromolecular-interaction mechanistic studies. Third, the accuracy of the measurements allows the application of more intricate interaction models than simple Langmuir monophasic binding. CONCLUSIONS: The detection and measurement of antibody binding by the type 1 diabetes autoantigen GAD65 represents an example of an antibody-antigen interaction where good structural, mechanistic and immunological data are available. Using SPRi we were able to characterise the kinetics of the interaction in greater detail than ELISA/RIA methods. Furthermore, our data indicate that SPRi is well suited to a multiplexed immunoassay using GAD65 proteins, and may be applicable to other biomarkers

Interaction of Aspirin (Acetylsalicylic Acid) with Lipid Membranes

We studied the interaction of Aspirin (acetylsalicylic acid) with lipid membranes using x-ray diffraction for bilayers containing up to 50 mol% of aspirin. From 2D x-ray intensity maps that cover large areas of reciprocal space we determined the position of the ASA molecules in the phospholipid bilayers and the molecular arrangement of the molecules in the plane of the membranes. We present direct experimental evidence that ASA molecules participate in saturated lipid bilayers of DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) and preferably reside in the head group region of the membrane. Up to 50 mol% ASA molecules can be dissolved in this type of bilayer before the lateral membrane organization is disturbed and the membranes are found to form an ordered, 2D crystal-like structure. Furthermore, ASA and cholesterol were found to co-exist in saturated lipid bilayers, with the ASA molecules residing in the head group region and the cholesterol molecules participating in the hydrophobic membrane core

Reverse Engineering the Yeast RNR1 Transcriptional Control System

Transcription is controlled by multi-protein complexes binding to short non-coding regions of genomic DNA. These complexes interact combinatorially. A major goal of modern biology is to provide simple models that predict this complex behavior. The yeast gene RNR1 is transcribed periodically during the cell cycle. Here, we present a pilot study to demonstrate a new method of deciphering the logic behind transcriptional regulation. We took regular samples from cell cycle synchronized cultures of Saccharomyces cerevisiae and extracted nuclear protein. We tested these samples to measure the amount of protein that bound to seven different 16 base pair sequences of DNA that have been previously identified as protein binding locations in the promoter of the RNR1 gene. These tests were performed using surface plasmon resonance. We found that the surface plasmon resonance signals showed significant variation throughout the cell cycle. We correlated the protein binding data with previously published mRNA expression data and interpreted this to show that transcription requires protein bound to a particular site and either five different sites or one additional sites. We conclude that this demonstrates the feasibility of this approach to decipher the combinatorial logic of transcription

Chemical Linkage to Injected Tissues Is a Distinctive Property of Oxidized Avidin

We recently reported that the oxidized avidin, named AvidinOX®, resides for weeks within injected tissues as a consequence of the formation of Schiff's bases between its aldehyde groups and tissue protein amino groups. We also showed, in a mouse pre-clinical model, the usefulness of AvidinOX for the delivery of radiolabeled biotin to inoperable tumors. Taking into account that AvidinOX is the first oxidized glycoprotein known to chemically link to injected tissues, we tested in the mouse a panel of additional oxidized glycoproteins, with the aim of investigating the phenomenon. We produced oxidized ovalbumin and mannosylated streptavidin which share with avidin glycosylation pattern and tetrameric structure, respectively and found that neither of them linked significantly to cells in vitro nor to injected tissues in vivo, despite the presence of functional aldehyde groups. The study, extended to additional oxidized glycoproteins, showed that the in vivo chemical conjugation is a distinctive property of the oxidized avidin. Relevance of the high cationic charge of avidin into the stable linkage of AvidinOX to tissues is demonstrated as the oxidized acetylated avidin lost the property. Plasmon resonance on matrix proteins and cellular impedance analyses showed in vitro that avidin exhibits a peculiar interaction with proteins and cells that allows the formation of highly stable Schiff's bases, after oxidation

Measures and Limits of Models of Fixation Selection

Models of fixation selection are a central tool in the quest to understand how the human mind selects relevant information. Using this tool in the evaluation of competing claims often requires comparing different models' relative performance in predicting eye movements. However, studies use a wide variety of performance measures with markedly different properties, which makes a comparison difficult. We make three main contributions to this line of research: First we argue for a set of desirable properties, review commonly used measures, and conclude that no single measure unites all desirable properties. However the area under the ROC curve (a classification measure) and the KL-divergence (a distance measure of probability distributions) combine many desirable properties and allow a meaningful comparison of critical model performance. We give an analytical proof of the linearity of the ROC measure with respect to averaging over subjects and demonstrate an appropriate correction of entropy-based measures like KL-divergence for small sample sizes in the context of eye-tracking data. Second, we provide a lower bound and an upper bound of these measures, based on image-independent properties of fixation data and between subject consistency respectively. Based on these bounds it is possible to give a reference frame to judge the predictive power of a model of fixation selection . We provide open-source python code to compute the reference frame. Third, we show that the upper, between subject consistency bound holds only for models that predict averages of subject populations. Departing from this we show that incorporating subject-specific viewing behavior can generate predictions which surpass that upper bound. Taken together, these findings lay out the required information that allow a well-founded judgment of the quality of any model of fixation selection and should therefore be reported when a new model is introduced

Preparation and Characterization of the Extracellular Domain of Human Sid-1

In C. elegans, the cell surface protein Sid-1 imports extracellular dsRNA into the cytosol of most non-neuronal cells, enabling systemic spread of RNA interference (RNAi) throughout the worm. Sid-1 homologs are found in many other animals, although for most a function for the protein has not yet been established. Sid-1 proteins are composed of an N-terminal extracellular domain (ECD) followed by 9–12 predicted transmembrane regions. We developed a baculovirus system to express and purify the ECD of the human Sid-1 protein SidT1. Recombinant SidT1 ECD is glycosylated and spontaneously assembles into a stable and discrete tetrameric structure. Electron microscopy (EM) and small angle x-ray scattering (SAXS) studies reveal that the SidT1 ECD tetramer is a compact, puck-shaped globular particle, which we hypothesize may control access of dsRNA to the transmembrane pore. These characterizations provide inroads towards understanding the mechanism of this unique RNA transport system from structural prospective

The Protein Partners of GTP Cyclohydrolase I in Rat Organs

GTP cyclohydrolase I (GCH1) is the rate-limiting enzyme for tetrahydrobiopterin biosynthesis and has been shown to be a promising therapeutic target in ischemic heart disease, hypertension, atherosclerosis and diabetes. The endogenous GCH1-interacting partners have not been identified. Here, we determined endogenous GCH1-interacting proteins in rat.A pulldown and proteomics approach were used to identify GCH1 interacting proteins in rat liver, brain, heart and kidney. We demonstrated that GCH1 interacts with at least 17 proteins including GTP cyclohydrolase I feedback regulatory protein (GFRP) in rat liver by affinity purification followed by proteomics and validated six protein partners in liver, brain, heart and kidney by immunoblotting. GCH1 interacts with GFRP and very long-chain specific acyl-CoA dehydrogenase in the liver, tubulin beta-2A chain in the liver and brain, DnaJ homolog subfamily A member 1 and fatty aldehyde dehydrogenase in the liver, heart and kidney and eukaryotic translation initiation factor 3 subunit I (EIF3I) in all organs tested. Furthermore, GCH1 associates with mitochondrial proteins and GCH1 itself locates in mitochondria.GCH1 interacts with proteins in an organ dependant manner and EIF3I might be a general regulator of GCH1. Our finding indicates GCH1 might have broader functions beyond tetrahydrobiopterin biosynthesis

Interaction of Rio1 Kinase with Toyocamycin Reveals a Conformational Switch That Controls Oligomeric State and Catalytic Activity

Rio1 kinase is an essential ribosome-processing factor required for proper maturation of 40 S ribosomal subunit. Although its structure is known, several questions regarding its functional remain to be addressed. We report that both Archaeoglobus fulgidus and human Rio1 bind more tightly to an adenosine analog, toyocamycin, than to ATP. Toyocamycin has antibiotic, antiviral and cytotoxic properties, and is known to inhibit ribosome biogenesis, specifically the maturation of 40 S. We determined the X-ray crystal structure of toyocamycin bound to Rio1 at 2.0 Å and demonstrated that toyocamycin binds in the ATP binding pocket of the protein. Despite this, measured steady state kinetics were inconsistent with strict competitive inhibition by toyocamycin. In analyzing this interaction, we discovered that Rio1 is capable of accessing multiple distinct oligomeric states and that toyocamycin may inhibit Rio1 by stabilizing a less catalytically active oligomer. We also present evidence of substrate inhibition by high concentrations of ATP for both archaeal and human Rio1. Oligomeric state studies show both proteins access a higher order oligomeric state in the presence of ATP. The study revealed that autophosphorylation by Rio1 reduces oligomer formation and promotes monomerization, resulting in the most active species. Taken together, these results suggest the activity of Rio1 may be modulated by regulating its oligomerization properties in a conserved mechanism, identifies the first ribosome processing target of toyocamycin and presents the first small molecule inhibitor of Rio1 kinase activity