Functional dynamics in cyclic nucleotide signaling and amyloid inhibition.

It is now established that understanding the molecular basis of biological function requires atomic resolution maps of both structure and dynamics. Here, we review several illustrative examples of functional dynamics selected from our work on cyclic nucleotide signaling and amyloid inhibition. Although fundamentally diverse, a central aspect common to both fields is that function can only be rationalized by considering dynamic equilibria between distinct states of the accessible free energy landscape. The dynamic exchange between ground and excited states of signaling proteins is essential to explain auto-inhibition and allosteric activation. The dynamic exchange between non-toxic monomeric species and toxic oligomers of amyloidogenic proteins provides a foundation to understand amyloid inhibition. NMR ideally probes both types of dynamic exchange at atomic resolution. Specifically, we will show how NMR was utilized to reveal the dynamical basis of cyclic nucleotide affinity, selectivity, agonism and antagonism in multiple eukaryotic cAMP and cGMP receptors. We will also illustrate how NMR revealed the mechanism of action of plasma proteins that act as extracellular chaperones and inhibit the self-association of the prototypical amyloidogenic Aß peptide. The examples outlined in this review illustrate the widespread implications of functional dynamics and the power of NMR as an indispensable tool in molecular pharmacology and pathology.

Aß association inhibition by transferrin.

The iron-transport glycoprotein transferrin has recently been shown to serve as a potent inhibitor of Aß self-association. Although this novel, to our knowledge, inhibitory function of transferrin is of potential therapeutic interest for the treatment of Alzheimer's disease, the underlying mechanism is still not fully understood. Although it has been shown that the Fe(III) sequestration by transferrin reduces oxidative damage and Aß aggregation, it is not clear whether transferrin is also able to inhibit Aß self-association through direct binding of Aß. Here, using saturation transfer and off-resonance relaxation NMR spectroscopy, we show that transferrin inhibits Aß aggregation also by preferentially binding Aß oligomers and outcompeting Aß monomers that would otherwise cause the growth of the Aß oligomers into larger assemblies. This inhibitory mechanism is different from the iron-sequestration model, but it is qualitatively similar to a mechanism previously proposed for the inhibition of Aß self-association by another plasma and cerebrospinal fluid protein, i.e., human serum albumin. These results suggest that Aß monomer competition through direct Aß oligomer binding might be a general strategy adopted by proteins in plasma and cerebrospinal fluid to prevent Aß aggregation.

Mapping the interactions between the Alzheimer's Aß-peptide and human serum albumin beyond domain resolution.

Human serum albumin (HSA) is a potent inhibitor of Aß self-association and this novel, to our knowledge, function of HSA is of potential therapeutic interest for the treatment of Alzheimer's disease. It is known that HSA interacts with Aß oligomers through binding sites evenly partitioned across the three albumin domains and with comparable affinities. However, as of this writing, no information is available on the HSA-Aß interactions beyond domain resolution. Here, we map the HSA-Aß interactions at subdomain and peptide resolution. We show that each separate subdomain of HSA domain 3 inhibits Aß self-association. We also show that fatty acids (FAs) compete with Aß oligomers for binding to domain 3, but the determinant of the HSA/Aß oligomer interactions are markedly distinct from those of FAs. Although salt bridges with the FA carboxylate determine the FA binding affinities, hydrophobic contacts are pivotal for Aß oligomer recognition. Specifically, we identified a site of Aß oligomer recognition that spans the HSA (494-515) region and aligns with the central hydrophobic core of Aß. The HSA (495-515) segment includes residues affected by FA binding and this segment is prone to self-associate into ß-amyloids, suggesting that sites involved in fibrilization may provide a lead to develop inhibitors of Aß self-association.

A sputum bioassay for airway eosinophilia using an eosinophil peroxidase aptamer.

Eosinophils are granulocytes that play a significant role in the pathogenesis of asthma and other airway diseases. Directing patient treatment based on the level of eosinophilia has been shown to be extremely effective in reducing exacerbations and therefore has tremendous potential as a routine clinical test. Herein, we describe the in vitro selection and optimization of DNA aptamers that bind to eosinophil peroxidase (EPX), a protein biomarker unique to eosinophils. Fifteen rounds of magnetic bead aptamer selection were performed prior to high throughput DNA sequencing. The top 10 aptamer candidates were assessed for EPX binding using a mobility shift assay. This process identified a lead aptamer candidate termed EAP1-05 with low nanomolar affinity and high specificity for EPX over other common sputum proteins. This aptamer sequence was further optimized through truncation and used to develop an easy-to-use colourimetric pull-down assay that can detect EPX over a concentration range from 1 - 100 nM in processed sputum. Forty-six clinical samples were processed using a new sputum dispersal method, appropriate for a rapid assessment assay, that avoids centrifugation and lengthy processing times. The assay showed 89% sensitivity and 96% specificity to detect eosinophilia (compared to gold standard sputum cytometry), with results being produced in under an hour. This assay could allow for an easy assessment of eosinophil activity in the airway to guide anti-inflammatory therapy for several airway diseases.

Stoichiometry and affinity of the human serum albumin-Alzheimer's Aß peptide interactions.

A promising strategy to control the aggregation of the Alzheimer's Aß peptide in the brain is the clearance of Aß from the central nervous system into the peripheral blood plasma. Among plasma proteins, human serum albumin plays a critical role in the Aß clearance to the peripheral sink by binding to Aß oligomers and preventing further growth into fibrils. However, the stoichiometry and the affinities of the albumin-Aß oligomer interactions are still to be fully characterized. For this purpose, here we investigate the Aß oligomer-albumin complexes through a novel and generally applicable experimental strategy combining saturation transfer and off-resonance relaxation NMR experiments with ultrafiltration, domain deletions, and dynamic light scattering. Our results show that the Aß oligomers are recognized by albumin through sites that are evenly partitioned across the three albumin domains and that bind the Aß oligomers with similar dissociation constants in the 1-100 nM range, as assessed based on a Scatchard-like model of the albumin inhibition isotherms. Our data not only explain why albumin is able to inhibit amyloid formation at physiological nM Aß concentrations, but are also consistent with the presence of a single high affinity albumin-binding site per Aß protofibril, which avoids the formation of extended insoluble aggregates.

Human serum albumin inhibits Abeta fibrillization through a "monomer-competitor" mechanism.

Human serum albumin (HSA) is not only a fatty acid and drug carrier protein, it is also a potent inhibitor of Abeta self-association in plasma. However, the mechanism underlying the inhibition of Abeta fibrillization by HSA is still not fully understood. We therefore investigated the Abeta-HSA system using a combined experimental strategy based on saturation transfer difference (STD) NMR and intrinsic albumin fluorescence experiments on three Abeta peptides with different aggregation propensities (i.e., Abeta(12-28), Abeta(1-40), and Abeta(1-42)). Our data consistently show that albumin selectively binds to cross-beta-structured Abeta oligomers as opposed to Abeta monomers. The HSA/Abeta oligomer complexes have K(D) values in the micromolar to submicromolar range and compete with the further addition of Abeta monomers to the Abeta assemblies, thus inhibiting fibril growth ("monomer competitor" model). Other putative mechanisms, according to which albumin acts as a "monomer stabilizer" or a "dissociation catalyst", are not supported by our data, thus resolving previous discrepancies in the literature regarding Abeta-HSA interactions. In addition, the model and the experimental approaches proposed here are anticipated to have broad relevance for the characterization of other systems that involve amyloidogenic peptides and oligomerization inhibitors.

Analysis and optimization of saturation transfer difference NMR experiments designed to map early self-association events in amyloidogenic peptides.

Saturation transfer difference (STD) methods recently have been proposed to be a promising tool for self-recognition mapping at residue and atomic resolution in amyloidogenic peptides. Despite the significant potential of the STD approach for systems undergoing oligomer/monomer (O/M) equilibria, a systematic analysis of the possible artifacts arising in this novel application of STD experiments is still lacking. Here, we have analyzed the STD method as applied to O/M peptides, and we have identified three major sources of possible biases: offset effects, intramonomer cross-relaxation, and partial spin-diffusion within the oligomers. For the purpose of quantitatively assessing these artifacts, we employed a comparative approach that relies on 1-D and 2-D STD data acquired at different saturation frequencies on samples with different peptide concentrations and filtration states. This artifact evaluation protocol was applied to the Abeta(12-28) model system, and all three types of artifacts appear to affect the measured STD spectra. In addition, we propose a method to minimize the biases introduced by these artifacts in the Halpha STD distributions used to obtain peptide self-recognition maps at residue resolution. This method relies on the averaging of STD data sets acquired at different saturation frequencies and provides results comparable to those independently obtained through other NMR pulse sequences that probe oligomerization, such as nonselective off-resonance relaxation experiments. The artifact evaluation protocol and the multiple frequencies averaging strategy proposed here are of general utility for the growing family of amyloidogenic peptides, as they provide a reliable analysis of STD spectra in terms of polypeptide self-recognition epitopes.

Analysis and parametric optimization of 1H off-resonance relaxation NMR experiments designed to map polypeptide self-recognition and other noncovalent interactions.

The measurement of 1H off-resonance nonselective relaxation rates (R(theta,ns)) has been recently proposed as an effective method to probe peptide self-recognition, opening new perspectives in the understanding of the prefibrillization oligomerization processes in amylodogenesis. However, a full analysis and parametric optimization of the NMR experiments designed to measure R(theta,ns) relaxation rates is still missing. Here we analyze the dependence of the R(theta,ns) rates upon three critical parameters: the tilt angle of the effective field during the spin lock, the static magnetic field, and finally the repetition delay. Our analysis reveals that the tilt angle theta = 35.5 degrees not only minimizes spin-diffusion, but also avoids experimental artifacts such as J-transfer and poor adiabaticity. In addition, we found that when the dominant relaxation mechanism is caused by uncorrelated pairwise 1H dipole-1H dipole interactions the R(35.5 degrees,ns) rate is not significantly affected by static field variations, suggesting a wide applicability of the 1H off-resonance nonselective relaxation experiment. Finally, we show that the self-recognition maps based on the comparative analysis of the R(35.5 degrees,ns) rates can tolerate decreases in the interscan delays without significantly compromising the identification of critical self-association loci. These considerations not only provide a better understanding of the 1H off-resonance nonselective relaxation, but they also serve as guidelines for the optimal setup of this experiment.

In vitro amyloid-ß binding and inhibition of amyloid-ß self-association by therapeutic albumin.

BACKGROUND: A promising approach for treating Alzheimer's disease relies on the net efflux of the amyloid-ß (Aß) peptide from the brain to peripheral plasma, as a result of plasma Aß clearance promoted by plasma removal and therapeutic albumin replacement. OBJECTIVE: To assess the binding of therapeutic albumin (Albutein, Grifols) to monomeric and aggregated Aß according to methods previously tested on the interactions between Aß and research-grade albumin. METHODS: Albumin integrity and the interactions with albumin stabilizers (octanoic acid and N-Ac-Trp) were assessed through one-dimensional (1D) 1H-NMR and saturation transfer difference (STD) NMR spectra. The interactions between monomeric Aß1-40 and albumin were probed by 2D 1H-15 N HSQC spectra of labeled Aß1-40. The formation of cross-ß structured Aß1-42 assemblies was monitored by ThT fluorescence. The interactions between self-assembled Aß1-42 and albumin were probed by Trp fluorescence. RESULTS: NMR spectra indicated that both therapeutic and research-grade albumin are similarly well-folded proteins. No significant changes in either HSQC peak position or intensity were observed upon addition of albumin to 15N-labeled Aß1-40, which rules out binding of albumin to monomeric Aß with dissociation constant in the µM or lower range. When aggregated Aß1-42 was added to albumin, quenching of Trp fluorescence was observed, which indicates albumin binding to Aß1-42 aggregates. The relative potency of therapeutic albumin as an Aß self-association inhibitor was in the same order of magnitude as research-grade albumin. CONCLUSIONS: Albutein inhibited Aß self-association by selectively binding Aß aggregates rather than monomers and by preventing further growth of the Aß assemblies.

A new function of human HtrA2 as an amyloid-beta oligomerization inhibitor.

Human HtrA2 is part of the HtrA family of ATP-independent serine proteases that are conserved in both prokaryotes and eukaryotes and localizes to the intermembrane space of the mitochondria. Several recent reports have suggested that HtrA2 is important for maintaining proper mitochondrial homeostasis and may play a role in Alzheimer's disease (AD), which is characterized by the presence of aggregates of the amyloid-beta peptide 1-42 (Abeta1-42). In this study, we analyzed the ability of HtrA2 to delay the aggregation of the model substrate citrate synthase (CS) and of the toxic Abeta1-42 peptide. We found that HtrA2 had a moderate ability to delay the aggregation of CS in vitro, and this activity was significantly enhanced when the PDZ domain was removed suggesting an inhibitory role for this domain on the activity. Additionally, using electron microscopy and nuclear magnetic resonance analyses, we observed that HtrA2 significantly delayed the aggregation of the Abeta1-42 peptide. Interestingly, the protease activity of HtrA2 and its PDZ domain were not essential for the delay of Abeta1-42 peptide aggregation. These results indicate that besides its protease activity, HtrA2 also performs a chaperone function and suggest a role for HtrA2 in the metabolism of intracellular Abeta and in AD.

Understanding the molecular basis for the inhibition of the Alzheimer's Abeta-peptide oligomerization by human serum albumin using saturation transfer difference and off-resonance relaxation NMR spectroscopy.

Human serum albumin (HSA) inhibits the formation of amyloid beta-peptide (Abeta) fibrils in human plasma. However, currently it is not known how HSA affects the formation of the highly toxic soluble diffusible oligomers that occur in the initial stages of Abeta fibrillization. We have therefore investigated by solution NMR the interaction of HSA with the Abeta(12-28) peptide, which has been previously shown to provide a reliable and stable model for the early prefibrillar oligomers as well as to contain key determinants for the recognition by albumin. For this purpose we propose a novel NMR approach based on the comparative analysis of Abeta in its inhibited and filtrated states monitored through both saturation transfer difference and recently developed nonselective off-resonance relaxation experiments. This combined NMR strategy reveals a mechanism for the oligomerization inhibitory function of HSA, according to which HSA targets preferentially the soluble oligomers of Abeta(12-28) rather than its monomeric state. Specifically, HSA caps the exposed hydrophobic patches located at the growing and/or transiently exposed sites of the Abeta oligomers, thereby blocking the addition of further monomers and the growth of the prefibrillar assemblies. The proposed model has implications not only for the pharmacological treatment of Alzheimer's disease specifically but also for the inhibition of oligomerization in amyloid-related diseases in general. In addition, the proposed NMR approach is expected to be useful for the investigation of the mechanism of action of other oligomerization inhibitors as well as of other amyloidogenic systems.