Quantitative Characterization of Metastability and Heterogeneity of Amyloid Aggregates

Amyloids are heterogeneous assemblies of extremely stable fibrillar aggregates of proteins. Although biological activities of the amyloids are dependent on its conformation, quantitative evaluation of heterogeneity of amyloids has been difficult. Here we use disaggregation of the amyloids of tetramethylrhodamine-labeled Aβ (TMR-Aβ) to characterize its stability and heterogeneity. Disaggregation of TMR-Aβ amyloids, monitored by fluorescence recovery of TMR, was negligible in native buffer even at low nanomolar concentrations but the kinetics increased exponentially with addition of denaturants such as urea or GdnCl. However, dissolution of TMR-Aβ amyloids is different from what is expected in the case of thermodynamic solubility. For example, the fraction of soluble amyloids is found to be independent of total concentration of the peptide at all concentrations of the denaturants. Additionally, soluble fraction is dependent on growth conditions such as temperature, pH, and aging of the amyloids. Furthermore, amyloids undissolved in a certain concentration of the denaturant do not show any further dissolution after dilution in the same solvent; instead, these require higher concentrations of the denaturant. Taken together, our results indicate that amyloids are a heterogeneous ensemble of metastable states. Furthermore, dissolution of each structurally homogeneous member requires a unique threshold concentration of denaturant. Fraction of soluble amyloids as a function of concentration of denaturants is found to be sigmoidal. The sigmoidal curve becomes progressively steeper with progressive seeding of the amyloids, although the midpoint remains unchanged. Therefore, heterogeneity of the amyloids is a major determinant of the steepness of the sigmoidal curve. The sigmoidal curve can be fit assuming a normal distribution for the population of the amyloids of various kinetic stabilities. We propose that the mean and the standard deviation of the normal distribution provide quantitative estimates of mean kinetic stability and heterogeneity, respectively, of the amyloids in a certain preparation.     

Dynamically Driven Ligand Selectivity in Cyclic Nucleotide Binding Domains

One of the mechanisms that minimize the aberrant cross-talk between cAMP- and cGMP-dependent signaling pathways relies on the selectivity of cAMP binding domains (CBDs). For instance, the CBDs of two critical eukaryotic cAMP receptors, i.e. protein kinase A (PKA) and the exchange protein activated by cAMP (EPAC), are both selectively activated by cAMP. However, the mechanisms underlying their cAMP versus cGMP selectivity are quite distinct. In PKA this selectivity is controlled mainly at the level of ligand affinity, whereas in EPAC it is mostly determined at the level of allostery. Currently, the molecular basis for these different selectivity mechanisms is not fully understood. We have therefore comparatively analyzed by NMR the cGMP-bound states of the essential CBDs of PKA and EPAC, revealing key differences between them. Specifically, cGMP binds PKA preserving the same syn base orientation as cAMP at the price of local steric clashes, which lead to a reduced affinity for cGMP. Unlike PKA, cGMP is recognized by EPAC in an anti conformation and generates several short and long range perturbations. Although these effects do not alter significantly the structure of the EPAC CBD investigated, remarkable differences in dynamics between the cAMP- and cGMP-bound states are detected for the ionic latch region. These observations suggest that one of the determinants of cGMP antagonism in EPAC is the modulation of the entropic control of inhibitory interactions and illustrate the pivotal role of allostery in determining signaling selectivity as a function of dynamic changes, even in the absence of significant affinity variations.In eukaryotes, protein kinase A (PKA)² and the exchange protein directly activated by cAMP (EPAC) are two major receptors for the cAMP second messenger (1–4). The activities of both PKA and EPAC are modulated in a cAMP-dependent manner through cAMP binding domains (CBDs) (1–4). In all isoforms of PKA, two tandem CBDs, denoted as CBD-A and CBD-B, are part of the regulatory subunit (R), in which they are preceded by an N-terminal dimerization docking module and a linker region (Fig. 1a) (1, 3). In the inactive state PKA exists as a tetrameric holo-enzyme complex, including two regulatory (R) subunits and two catalytic (C) subunits (1, 3). Binding of cAMP to the CBDs of the R subunits results in the release of the C subunits and in the activation of the kinase function (1, 3).Open in a separate windowFIGURE 1.Schematic representation of the domain organization in the regulatory subunit I-α of PKA (a) and in EPAC (b).The black circles indicate cAMP. a, D/D is the dimerization docking domain; the inhibitory site is shown in orange, and the two tandem cAMP binding domains, CBD-A and CBD-B, are highlighted in different shades of green. b, DEP, disheveled-egl-10-pleckstrin domain; REM, Ras exchange motif; RA, Ras-associated module, and the CDC25HD catalytic domain are represented in gray, green, orange, blue, and yellow, respectively. The black dashed line and the empty cAMP circle in EPAC2 indicate that this domain is not strictly necessary for the cAMP-dependent GEF activity. The module with question mark in EPAC1 denotes an unknown function for this domain. a and b, the CBD in light green shown below the full-length protein represents the construct used for the NMR studies. c, sequence alignment of the CBDs of bovine RIα- domain A and human EPAC1. Fully conserved residues are highlighted in green; cyan denotes conservation for the functional group only, and yellow indicates the residues present only in one of the CBDs. The secondary structure of apo-EPAC2_m (PDB ID 1O7F) is shown in red.Unlike PKA, EPAC is a single-chain protein that functions as a guanine nucleotide-exchange factor (GEF) for the small GTPase Rap1 and Rap2 (2, 4). The domain organization of EPAC includes an N-terminal regulatory region (RR) and a C-terminal catalytic region (CR) (Fig. 1b). There are two known homologous isoforms of EPAC, i.e. EPAC1 and EPAC2. One of the key differences between EPAC1 and EPAC2 is that in the former there is only a single CBD, whereas in the latter there are two noncontiguous CBDs, i.e. CBD-A and CBD-B. However, CBD-A has been shown not to be strictly necessary for the cAMP-dependent activation of EPAC (2, 4).Both PKA and EPAC are critical for the regulation of a wide range of cAMP-dependent physiological processes (1–4), and impaired activity of these cAMP sensors has been implicated in cardiovascular pathology, diabetes, and Alzheimer disease (1–4). Therefore, PKA and EPAC represent attractive therapeutic targets. However, the design and development of specific drug leads targeting the CBDs of either of these two eukaryotic protein systems require an in depth analysis of how PKA and EPAC selectively recognize and allosterically respond to diverse cNMPs. For instance, the cAMP and cGMP second messengers control distinct groups of essential signaling pathways (1–6). It is therefore critical to minimize the cross-talk between the cAMP- and cGMP-dependent cellular responses. Although in vivo the selective control of the cAMP- and/or cGMP-dependent signaling pathways is a complex process that depends on multiple factors, including the modulation of cNMP synthesis, degradation, and compartmentalization (5), one of the key mechanisms to reduce the cAMP/cGMP cross-talk relies on the ability of both PKA and EPAC CBDs to sense selectively cAMP as opposed to cGMP.Despite the fact that both PKA and EPAC CBDs are cAMP-selective sensors, these two signaling systems adopt different mechanisms to implement their cAMP-selective response. Specifically, in the PKA system cGMP is an agonist of cAMP, i.e. cGMP is able to activate PKA once it binds the R subunit. However, the affinity of cGMP for the PKA R subunit is significantly lower than that of cAMP, resulting in an activation constant that is 2 orders of magnitude higher than that of cAMP (i.e. K_a of 21 ± 2 nm for cAMP and of 4100 ± 20 nm for cGMP) (7, 8). Unlike PKA, EPAC preserves approximate micromolar affinities for both cAMP and cGMP, but in EPAC the latter cNMP is an antagonist of cAMP (9, 10), i.e. cGMP, like other N⁶-substituted cAMP analogs, binds effectively to the CBD of EPAC but fails to fully activate its GEF activity (9, 10).The molecular basis for the cGMP antagonism selectively observed in EPAC but not in PKA is currently not fully understood. An initial hypothesis to explain the antagonist function of cGMP in EPAC has been recently proposed based on the structure of the ternary (S_p)-cAMPS-EPAC2_m-Rap1 complex (11), which shows that the N⁶ of the (S_p)-cAMPS agonist forms an hydrogen-bond with the backbone carbonyl oxygen of Lys-450 located in the lid region (supplemental Table S1) (11). The disruption of this hydrogen bond by cGMP has been hypothesized to result in the inhibition of EPAC activation (11). However, a similar backbone hydrogen bond between the N⁶ of cAMP and the backbone carbonyl oxygen of Arg-632 has been observed also in the CBDs of the hyperpolarization-activated cyclic nucleotide-modulated channels (HCN) (supplemental Table S1), for which cGMP, like in PKA, is not an antagonist (12). This observation suggests that the elimination of the cAMP N⁶ hydrogen bond alone may not be sufficient to fully explain why cGMP is a cAMP antagonist with respect to the activation of EPAC. Furthermore, the previously proposed hypothesis based on the simple disruption by cGMP of the N⁶ hydrogen bond does not consider the possibility that cGMP may adopt an EPAC-bound conformation different from that of cAMP and/or that cGMP may affect also the inhibitory interactions between the ionic latch residues of the EPAC RR and the CDC25HD catalytic domain. These RR/CR salt bridges stabilize the EPAC system in an overall “closed” topology, whereby the RR sterically occludes access of Rap1 into the catalytic domain of EPAC (13). cAMP binding results in increased picosecond to nanosecond and millisecond to microsecond dynamics at the ionic latch region, which in turn leads to an increased entropic penalty for the inhibitory interactions mediated by the ionic latch (14). In other words, cAMP is able to weaken the inhibitory interactions between the regulatory and catalytic regions of EPAC by increasing the entropic cost associated with the formation of the cluster of ionic latch salt bridges between these two functional segments. This dynamically driven mechanism contributes to the observed cAMP-dependent shift toward active “open” conformations of EPAC, and we hypothesize that one of the effects of cGMP is to perturb the dynamic patterns of the EPAC CBD, thus altering the entropic control of the inhibitory interactions.To test our hypotheses on cGMP agonism/antagonism and to further understand the molecular mechanisms underlying the different signaling responses of PKA and EPAC to the two endogenous second messengers cAMP and cGMP, here we present a comparative NMR analysis of cGMP binding and allostery for the critical CBDs of both PKA and EPAC. These results were also compared with the data on the same domains in their apo- and cAMP-bound states (14–20). All these studies rely on the RIα-(119–244) and the related EPAC1_h-(149–318) constructs (Fig. 1), which have been previously validated as models of the essential CBDs of PKA and EPAC, respectively (14–20).Our comparative NMR analysis has revealed that the structure, dynamics, and allosteric activation pathways of the PKA CBD-A are not significantly altered when cAMP is replaced by cGMP. However, significant differences between these two cNMPs are found for the EPAC1_h CBD at the level of both ligand recognition and modulation of dynamic modes, leading to a mechanism in which cGMP shifts the activation equilibrium of EPAC toward the auto-inhibited state, thus accounting for its antagonistic function.     

Conformational change in rhomboid protease GlpG induced by inhibitor binding to its S' subsites

Rhomboid protease conducts proteolysis inside the hydrophobic environment of the membrane. The conformational flexibility of the protease is essential for the enzyme mechanism, but the nature of this flexibility is not completely understood. Here we describe the crystal structure of rhomboid protease GlpG in complex with a phosphonofluoridate inhibitor, which is covalently bonded to the catalytic serine and extends into the S' side of the substrate binding cleft. Inhibitor binding causes subtle but extensive changes in the membrane protease. Many transmembrane helices tilt and shift positions, and the gap between S2 and S5 is slightly widened so that the inhibitor can bind between them. The side chain of Phe-245 from a loop (L5) that acts as a cap rotates and uncovers the opening of the substrate binding cleft to the lipid bilayer. A concurrent turn of the polypeptide backbone at Phe-245 moves the rest of the cap and exposes the catalytic serine to the aqueous solution. This study, together with earlier crystallographic investigation of smaller inhibitors, suggests a simple model for explaining substrate binding to rhomboid protease.     

Structural differences between apolipoprotein E3 and E4 as measured by 19F NMR

The apolipoprotein E family contains three major isoforms (ApoE4, E3, and E2) that are directly involved with lipoprotein metabolism and cholesterol transport. ApoE3 and apoE4 differ in only a single amino acid with an arginine in apoE4 changed to a cysteine at position 112 in apoE3. Yet only apoE4 is recognized as a risk factor for Alzheimer''s disease. Here we used ¹⁹F NMR to examine structural differences between apoE4 and apoE3 and the effect of the C-terminal domain on the N-terminal domain. After incorporation of 5-¹⁹F-tryptophan the 1D ¹⁹F NMR spectra were compared for the N-terminal domain and for the full length proteins. The NMR spectra of the N-terminal region (residues 1–191) are reasonably well resolved while those of the full length wild-type proteins are broad and ill-defined suggesting considerable conformational heterogeneity. At least four of the seven tryptophan residues in the wild type protein appear to be solvent exposed. NMR spectra of the wild-type proteins were compared to apoE containing four mutations in the C-terminal region that gives rise to a monomeric form either of apoE3 under native conditions (Zhang et al., Biochemistry 2007; 46: 10722–10732) or apoE4 in the presence of 1 M urea. For either wild-type or mutant proteins the differences in tryptophan resonances in the N-terminal region of the protein suggest structural differences between apoE3 and apoE4. We conclude that these differences occur both as a consequence of the Arg158Cys mutation and as a consequence of the interaction with the C-terminal domain.     

Dissociation of apolipoprotein E oligomers to monomer is required for high-affinity binding to phospholipid vesicles

The apolipoprotein apoE plays a key role in cholesterol and lipid metabolism. There are three isoforms of this protein, one of which, apoE4, is the major risk factor for Alzheimer's disease. At micromolar concentrations all lipid-free apoE isoforms exist primarily as monomers, dimers, and tetramers. However, the molecular weight form of apoE that binds to lipid has not been clearly defined. We have examined the role of self-association of apoE with respect to interactions with phospholipids. Measurements of the time dependence of turbidity clearance of small unilamellar vesicles of dimyristoyl-sn-glycero-3-phosphocholine (DMPC) upon addition of apoE show that higher molecular weight oligomers bind poorly if at all. The kinetic data can be described by a reaction model in which tetramers and dimers of apoE must first dissociate to monomers which then bind to the liposome surface in a fast and reversible manner. A slow but not readily reversible conformational conversion of the monomer then occurs. Prior knowledge of the rate constants for the association-dissociation process allows us to determine the rate constant of the conformational conversion. This rate constant is isoform dependent and appears to correlate with the stability of the apoE isoforms with the rate of dissociation of the apoE oligomers to monomers being the rate-limiting process for lipidation. Differences in the lipidation kinetics between the apoE isoforms arise from their differences in the self-association behavior leading to the conclusion that self-association behavior may influence biological functions of apoE in an isoform-dependent manner.     

Concerning the structure of apoE

Apolipoprotein E (apoE), first described in 1973, is a truly fascinating protein. While studies initially focused on its role in cholesterol and lipid metabolism, one apoE isoform (apoE4) is a major risk factor for development of late onset Alzheimer's disease. Yet the difference between apoE3, the common form, and apoE4 is a single amino acid of the 299 in this 34 kDa protein. Structure determination of the two domain full length apoE3 protein was only accomplished in 2011 and supports the notion that mutations in the N‐terminal domain can be propagated through the structure to the C‐terminal domain. Understanding the structural differences between apoE3 and apoE4 is critical for finding ways to modulate the deleterious effect of apoE4.     

Bacopasaponins E and F: two jujubogenin bisdesmosides from Bacopa monniera

Two new dammarane-type jujubogenin bisdesmosides, bacopasaponins E and F of biological interest have been isolated from the reputed Indian medicinal plant Bacopa monniera and defined as 3-O-[beta-D-glucopyranosyl(1 --> 3)[alpha-L-arabinofuranosyl(1 --> 2)]alpha-L-arabinopyranosyl]-20-O-(alpha-L-arabinopyranosyl) jujubogenin and 3-O-[beta-D-glucopyranosyl(1 --> 3)[alpha-L-arabinofuranosyl(1 --> 2)]beta-D-glucopyranosyl]-20-O-alpha-L-arabinopyranosyl) jujubogenin respectively by spectroscopic methods and some chemical transformations.     

Immunomodulatory effects of two sapogenins 1 and 2 isolated from Luffa cylindrica in Balb/C mice

Two Triterpenoids (sapogenins 1 and 2) isolated from Luffa cylindrica were subjected to immunomodulatory activity in male Balb/c mice. Mice were treated with three doses of sapogenins 1 and 2 (10, 30 and 100 mg/kg) and levamisole (2.5 mg/kg) used as a standard reference drug for 15 days. Immune responses to T-dependent antigen SRBCs were observed using parameters like HA, PFC, DTH, lymphocyte proliferation and phagocytosis. As regards these parameters, sapogenins 1 and 2 elicited a significant increase in the HA, PFC and DTH response at dose 10 mg/kg (P<0.01) and 100 mg/kg (P<0.001), respectively. Sapogenins 1 and 2 also showed significant dose-dependent decrease and increase in lymphocyte proliferation assay and phagocytic activity of macrophages. Overall, sapogenins 1 and 2 showed dose relative immunostimulatory effect on in vivo immune functions in mice.