Calmodulin Transduces Ca²⁺ Oscillations into Differential Regulation of Its Target Proteins

Diverse physiological processes are regulated differentially by Ca²⁺ oscillations through the common regulatory hub calmodulin. The capacity of calmodulin to combine specificity with promiscuity remains to be resolved. Here we propose a mechanism based on the molecular properties of calmodulin, its two domains with separate Ca²⁺ binding affinities, and target exchange rates that depend on both target identity and Ca²⁺ occupancy. The binding dynamics among Ca²⁺, Mg²⁺, calmodulin, and its targets were modeled with mass-action differential equations based on experimentally determined protein concentrations and rate constants. The model predicts that the activation of calcineurin and nitric oxide synthase depends nonmonotonically on Ca²⁺-oscillation frequency. Preferential activation reaches a maximum at a target-specific frequency. Differential activation arises from the accumulation of inactive calmodulin-target intermediate complexes between Ca²⁺ transients. Their accumulation provides the system with hysteresis and favors activation of some targets at the expense of others. The generality of this result was tested by simulating 60 000 networks with two, four, or eight targets with concentrations and rate constants from experimentally determined ranges. Most networks exhibit differential activation that increases in magnitude with the number of targets. Moreover, differential activation increases with decreasing calmodulin concentration due to competition among targets. The results rationalize calmodulin signaling in terms of the network topology and the molecular properties of calmodulin

Fluorescent Filter-Trap Assay for Amyloid Fibril Formation Kinetics in Complex Solutions

Amyloid fibrils are the most distinct components of the plaques associated with various neurodegenerative diseases. Kinetic studies of amyloid fibril formation shed light on the microscopic mechanisms that underlie this process as well as the contributions of internal and external factors to the interplay between different mechanistic steps. Thioflavin T is a widely used noncovalent fluorescent probe for monitoring amyloid fibril formation; however, it may suffer from limitations due to the unspecific interactions between the dye and the additives. Here, we present the results of a filter-trap assay combined with the detection of fluorescently labeled amyloid β (Aβ) peptide. The filter-trap assay separates formed aggregates based on size, and the fluorescent label attached to Aβ allows for their detection. The times of half completion of the process (<i>t</i>_1/2) obtained by the filter-trap assay are comparable to values from the ThT assay. High concentrations of human serum albumin (HSA) and carboxyl-modified polystyrene nanoparticles lead to an elevated ThT signal, masking a possible fibril formation event. The filter-trap assay allows fibril formation to be studied in the presence of those substances and shows that Aβ fibril formation is kinetically inhibited by HSA and that the amount of fibrils formed are reduced. In contrast, nanoparticles exhibit a dual-behavior governed by their concentration

Effects of Polyamino Acids and Polyelectrolytes on Amyloid β Fibril Formation

The fibril formation of the neurodegenerative peptide amyloid β (Aβ42) is sensitive to solution conditions, and several proteins and peptides have been found to retard the process. Aβ42 fibril formation was followed with ThT fluorescence in the presence of polyamino acids (poly-glutamic acid, poly-lysine, and poly-threonine) and other polymers (poly­(acrylic acid), poly­(ethylenimine), and poly­(diallyldimethylammonium chloride). An accelerating effect on the Aβ42 aggregation process is observed from all positively charged polymers, while no effect is seen from the negative or neutral polymers. The accelerating effect is dependent on the concentration of positive polymer in a highly reproducible manner. Acceleration is observed from a 1:500 polymer to Aβ42 weight ratio and up. Polyamino acids and the other polymers exert quantitatively the same effect at the same concentrations based on weight. Fibrils are formed in all cases as verified by transmission electron microscopy. The concentrations of polymers required for acceleration are too low to affect the Aβ42 aggregation process through increased ionic strength or molecular crowding effects. Instead, the acceleration seems to arise from the locally increased Aβ42 concentration near the polymers, which favors association and affects the electrostatic environment of the peptide

Binding of Human C4BP to S. pyogenes Strains of Different M Types

<p>Strains of the M types indicated were analyzed for ability to bind radiolabelled C4BP. Upper panel: OF⁺ strains. Lower panel: OF⁻ strains. Only strains that bound Fg, as determined in parallel tests, were used for the analysis because binding of Fg is a characteristic property of S. pyogenes isolates expressing members of the M protein family. Binding is expressed as percent of added radioactivity. The threshold for binding of C4BP was set at ≥10% binding (dashed line). Background binding to an M-negative strain (~3%) has been subtracted. All strains were tested at least twice, with duplicate samples, and the results were highly reproducible. For each strain, data from one experiment are shown. The data include binding data for one allelic variant per M type, except that data for both M4 and M4.1 are included. For strains of some M types, several allelic variants were tested and in most cases these strains did not differ in ability to bind C4BP (unpublished data).</p

Sequence Analysis of C4BP-Binding HVRs and Site-Specific Mutagenesis in M22

<div><p>(A) Sequence logo of C4BP-binding HVRs. The logo was generated from the seven C4BP-binding HVRs aligned in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020047#ppat-0020047-g001" target="_blank">Figure 1</a>B, using WebLogo (<a href="http://weblogo.berkeley.edu" target="_blank">http://weblogo.berkeley.edu</a>). Only regions in the HVRs corresponding to the shortest known C4BP-binding region in M22 (residues 1–39) were used to create the logo. These regions are demarcated by the vertical hatched lines in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020047#ppat-0020047-g001" target="_blank">Figure 1</a>B. In the logo, each column in the alignment is represented by a stack of letters, with the height of each letter proportional to the observed frequency of the corresponding residue at that position, while the overall height of each stack is proportional to the sequence conservation at that position [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020047#ppat-0020047-b063" target="_blank">63</a>]. The sequence of the M4.1 HVR was included in the generation of the logo, although it is virtually identical to M4, because the single residue difference between these two HVRs was important for the conclusion that the different HVRs completely lack residue identities (see text). The numbering below the logo refers to residue numbers in the M22 protein and putative coiled-coil heptads (a–g) in M22 are indicated. Asterisks show the position of four M22 residues (L21, E24, L28, and E31) analyzed by site-specific mutagenesis.</p><p>(B) Helical wheel representation of a dimeric coiled-coil [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020047#ppat-0020047-b042" target="_blank">42</a>]. The sequence of the L21–E31 region of M22 is included, with asterisks above residues L21, E24, L28, and E31, which were analyzed by site-specific mutagenesis and are located within the predicted coiled-coil region. The positions of residues within putative coiled-coil heptads (a–g) are indicated.</p><p>(C–E) The four mutant M22 proteins indicated, constructed by site-specific mutagenesis, and the wild-type (wt) M22 protein, were expressed in <i>S. pyogenes,</i> and the strains were analyzed for surface expression of the proteins and ability to bind C4BP. The genes encoding the proteins were present on plasmid pLZ12Spec, carried by an M-negative strain. This M-negative strain also served as negative control. (C) and (D) show that the different proteins were expressed normally on the bacterial surface (see text). (E) shows that mutants L21A and L28A had completely lost C4BP-binding ability, while mutants E24A and E31A were unaffected. The results shown in (C–E) are based on three separate experiments with duplicate samples and are presented as means ± SD.</p></div

Characterization of the C4BP-Binding HVR in M114

<div><p>(A) The HVR of M114 is a distinct protein domain that binds C4BP with high specificity. A dimerized synthetic peptide, derived from the 52 N-terminal residues in M114 and designated M114-N, was immobilized in a column. Whole human serum was passed through the column, which was washed and eluted. Control columns contained the C4BP-binding M22-N peptide or the nonbinding M5-N peptide. The eluates, and human serum, were analyzed by SDS-PAGE, as indicated. The ~70 kDa polypeptide present in the eluates from the M22-N and M114-N columns was identified as the C4BP α-chain by Western blot analysis with specific antiserum (not shown).</p><p>(B) The M114 and M22 proteins bind to the same region in C4BP. The peptides indicated were used to inhibit the binding of radiolabelled M22 protein to C4BP immobilized in microtiter wells. Data from three separate experiments with duplicate samples, presented as means ± SD.</p></div

Charge Dependent Retardation of Amyloid β Aggregation by Hydrophilic Proteins

The aggregation of amyloid β peptides (Aβ) into amyloid fibrils is implicated in the pathology of Alzheimer’s disease. In light of the increasing number of proteins reported to retard Aβ fibril formation, we investigated the influence of small hydrophilic model proteins of different charge on Aβ aggregation kinetics and their interaction with Aβ. We followed the amyloid fibril formation of Aβ40 and Aβ42 using thioflavin T fluorescence in the presence of six charge variants of calbindin D_9k and single-chain monellin. The formation of fibrils was verified with transmission electron microscopy. We observe retardation of the aggregation process from proteins with net charge +8, +2, −2, and −4, whereas no effect is observed for proteins with net charge of −6 and −8. The single-chain monellin mutant with the highest net charge, scMN+8, has the largest retarding effect on the amyloid fibril formation process, which is noticeably delayed at as low as a 0.01:1 scMN+8 to Aβ40 molar ratio. scMN+8 is also the mutant with the fastest association to Aβ40 as detected by surface plasmon resonance, although all retarding variants of calbindin D_9k and single-chain monellin bind to Aβ40

The HVRs of M4.1 and M114 Bind C4BP

<div><p>Fusion proteins, derived from the HVR of M4.1 or M114 and the C-terminal part of M5, were expressed in the M-negative strain S. pyogenes ΔM5 using genes carried on plasmid pLZ12Spec. Controls included a strain expressing the C4BP-binding fusion protein M22⁵⁷–M5, a strain expressing the non–C4BP-binding M5 protein, and the M-negative strain ΔM5.</p><p>(A) Surface expression analyzed with rabbit antibodies directed against the C-repeat region of M5. Bound antibodies were detected with radiolabelled protein A. Binding of protein A to the M22⁵⁷–M5 strain, incubated with antiserum diluted ×10², was defined as 100%. The M5-negative strain ΔM5 served as negative control.</p><p>(B) Binding of radiolabelled C4BP. Binding at the highest bacterial concentration to the control expressing M22⁵⁷–M5 was defined as 100%. The non–C4BP-binding M5 strain served as negative control. All data in (A) and (B) are based on three separate experiments with duplicate samples, and are presented as means ± SD.</p></div

Single Amino Acid Changes Not Affecting C4BP Binding Cause Major Antigenic Changes in the HVR of M22

<div><p>(A) Schematic representation of an inhibition test used to analyze the antigenic properties of mutant M22 proteins expressed on the surface of <i>S. pyogenes.</i> The test was based on the binding of mouse anti–M22-N to pure M22 protein immobilized in microtiter wells. This binding was inhibited with whole S. pyogenes bacteria expressing mutant M22 proteins.</p><p>(B) Ability of S. pyogenes strains expressing the M22 mutants E24A and E31A to cause inhibition. The positive control expressed the wild-type M22 protein and the negative control lacked M protein. As compared to the positive control, 50% inhibition (dashed line) required ~30-fold more bacteria expressing either of the mutant proteins. Results based on three separate experiments with duplicate samples, presented as means ± SD.</p></div