Artificial Antennas: Thermodynamics of Protein-DNA, Protein-Solid, and Protein-Dye Interactions for Light Harvesting Applications

The thermodynamics of protein-DNA and protein-solid interactions have been investigated here. Protein-DNA interactions play fundamental roles in biological systems and disease. Therefore, studies that help explain the mechanisms of these interactions will contribute to the development of much needed drug therapies. Protein-solid interactions have been optimized for applications in drug delivery and biomedical devices. Additionally, protein-DNA and protein-solid interactions have been optimized as matrices for artificial light harvesting antennas with the goal of utilizing sunlight for energy conversion. In this thesis, glucose oxidase/DNA (GOx/DNA) was used as a model system to study the role of protein surface charge in the thermodynamics of protein-DNA interactions. Synthesis of differentially charged GOx analogs facilitated control of its net charge and revealed a protein/DNA switching mechanism where binding is switched on at a GOx charge of +30. Another goal of this thesis was to study the contribution of protein surface charge to the thermodynamics of protein/solid interactions using a GOx/zirconium phosphate (GOx/α-ZrP) model system. Negatively charged GOx analogs associated weakly with α-ZrP but positively charged analogs associated with high affinity and there was a significant linear relationship between GOx net charge and GOx/α-ZrP binding affinity. In a third study, another protein, bovine serum albumin (BSA) was incorporated into a BSA/DNA matrix. A biodegradable BSA/DNA/dyes antenna that harvested light in the broad range from 350 nm to 590 nm was synthesized by self-assembly. Cascade energy transfer that shuttled photons to a terminal acceptor emitting red light for the potential catalysis of solar cells was characterized

Protective Effects of Positive Lysosomal Modulation in Alzheimer's Disease Transgenic Mouse Models

Alzheimer's disease (AD) is an age-related neurodegenerative pathology in which defects in proteolytic clearance of amyloid β peptide (Aβ) likely contribute to the progressive nature of the disorder. Lysosomal proteases of the cathepsin family exhibit up-regulation in response to accumulating proteins including Aβ1–42. Here, the lysosomal modulator Z-Phe-Ala-diazomethylketone (PADK) was used to test whether proteolytic activity can be enhanced to reduce the accumulation events in AD mouse models expressing different levels of Aβ pathology. Systemic PADK injections in APPSwInd and APPswe/PS1ΔE9 mice caused 3- to 8-fold increases in cathepsin B protein levels and 3- to 10-fold increases in the enzyme's activity in lysosomal fractions, while neprilysin and insulin-degrading enzyme remained unchanged. Biochemical analyses indicated the modulation predominantly targeted the active mature forms of cathepsin B and markedly changed Rab proteins but not LAMP1, suggesting the involvement of enhanced trafficking. The modulated lysosomal system led to reductions in both Aβ immunostaining as well as Aβx-42 sandwich ELISA measures in APPSwInd mice of 10–11 months. More extensive Aβ deposition in 20-22-month APPswe/PS1ΔE9 mice was also reduced by PADK. Selective ELISAs found that a corresponding production of the less pathogenic Aβ1–38 occurs as Aβ1–42 levels decrease in the mouse models, indicating that PADK treatment leads to Aβ truncation. Associated with Aβ clearance was the elimination of behavioral and synaptic protein deficits evident in the two transgenic models. These findings indicate that pharmacologically-controlled lysosomal modulation reduces Aβ1–42 accumulation, possibly through intracellular truncation that also influences extracellular deposition, and in turn offsets the defects in synaptic composition and cognitive functions. The selective modulation promotes clearance at different levels of Aβ pathology and provides proof-of-principle for small molecule therapeutic development for AD and possibly other protein accumulation disorders

Tuning Enzyme/α-Zr(IV) Phosphate Nanoplate Interactions via Chemical Modification of Glucose Oxidase

Using glucose oxidase (GOx) and α-Zr­(IV) phosphate nanoplates (α-ZrP) as a model system, a generally applicable approach to control enzyme–solid interactions via chemical modification of amino acid side chains of the enzyme is demonstrated. Net charge on GOx was systematically tuned by appending different amounts of polyamine to the protein surface to produce chemically modified GOx­(<i>n</i>), where <i>n</i> is the net charge on the enzyme after the modification and ranged from −62 to +95 electrostatic units in the system. The binding of GOx­(<i>n</i>) with α-ZrP nanosheets was studied by isothermal titration calorimetry (ITC) as well as by surface plasmon resonance (SPR) spectroscopy. Pristine GOx showed no affinity for the α-ZrP nanosheets, but GOx­(<i>n</i>) where <i>n</i> ≥ −20 showed binding affinities exceeding (2.1 ± 0.6) × 10⁶ M^–1, resulting from the charge modification of the enzyme. A plot of GOx­(<i>n</i>) charge vs Gibbs free energy of binding (Δ<i>G</i>) for <i>n</i> = +20 to <i>n</i> = +65 indicated an overall increase in favorable interaction between GOx­(<i>n</i>) and α-ZrP nanosheets. However, Δ<i>G</i> is less dependent on the net charge for <i>n</i> > +45, as evidenced by the decrease in the slope as charge increased further. All modified enzyme samples and enzyme/α-ZrP complexes retained a significant amount of folding structure (examined by circular dichroism) as well as enzymatic activities. Thus, strong control over enzyme–nanosheet interactions via modulating the net charge of enzymes may find potential applications in biosensing and biocatalysis

PADK selectively enhances cathepsin B levels in two transgenic mouse models.

<p>APP_SwInd and APP-PS1 mice were injected i.p. daily with PADK (20 mg/kg; n = 11−13) or vehicle (n = 10) for 9–11 days. Hippocampal homogenates were analyzed by immunoblot and mean immunoreactivities are shown for active cathepsin B (CB), neprilysin (nep), insulin-degrading enzyme (IDE), α-secretase (α-sec), and LAMP1.</p><p>***<i>P</i><0.0001, unpaired t-test.</p

PADK reduces behavioral deficits in APP_SwInd and APP-PS1 mice.

<p>In the first model, vehicle-treated wildtype mice (n = 11) were tested with groups of vehicle- (n = 10) and PADK-treated APP_SwInd mice (n = 13) across trials on the suspended rod test (A), and time maintained on the rod during the third trial was plotted (means±SEM). The animal groups were also tested across consecutive days in the same novel field, and the percent change±SEM in exploratory distance on the second day compared to the first was determined (B). In the second model, age-matched vehicle-treated wildtypes were tested with groups of vehicle- (n = 10) and PADK-treated APP-PS1 mice (n = 11) for spontaneous alternation behavior in a T-maze (C); data are plotted as percent of maximum alternations possible (mean±SEM). Open field mobility assessment confirmed no change in mean grid crossings±SEM across the three groups of mice (D). Post hoc tests compared to vehicle-treated transgenics: *<i>P</i>≤0.01, **<i>P</i><0.001.</p

PADK decreases intra- and extracellular 6E10 staining in APPswe/PS1ΔE9 (APP-PS1) mice of 20–22 months.

<p>The APP-PS1 mice received 11 daily injections of PADK (i.p., 20 mg/kg; n = 11) or vehicle (veh; n = 10), and non-transgenic control mice (wt) received vehicle injections. Fixed brain sections from the different groups were hematoxylin-eosin stained (A; arrows denote typical deposits) and 6E10 immunolabeled (B), indicating that PADK treatment reduces intra- and extracellular deposition in hippocampus. Equal protein samples from vehicle- (–) and PADK-treated (+) APP-PS1 mouse brains were immunoblotted with 6E10 antibody to assess the 4-kDa Aβ peptide and the parent hAPP, and with selective antibodies to label sAPPα and sAPPβ (C). Mean integrated optical densities±SEM for the different species were normalized to 100% as shown. The same brain samples were also tested by Aβ_x-42 sandwich ELISA to determine femtomoles of peptide per milligram protein (D). ANOVA: <i>P</i><0.0001; post hoc test compared to APP-PS1+vehicle: **<i>P</i><0.001. Unpaired t-test: *<i>P</i><0.01. Size bar: 400 µm, A; 50 µm, B. DG, dentate gyrus; sp, stratum pyramidale; sr, stratum radiatum.</p

Reduced intracellular Aβ_1–42 staining corresponds with enhanced cathepsin B.

<p>Fixed brain sections from vehicle-treated wildtype mice (wt) and from the APP-PS1 mice treated with vehicle (veh) or PADK were double-stained for Aβ_1–42 (green) and cathepsin B (red). Immunofluorescence images of CA1 pyramidal neurons (arrows) are shown, with view-field widths of 56 µm.</p

PADK has no inhibitory effect on β-secretase activity.

<p>Recombinant human β-secretase (10 ng/ml) was incubated with different concentrations of PADK (open triangles), CA074me (circles), and β-secretase inhibitor IV (closed triangles), and the enzyme activity was determined with the SensiZyme assay kit that uses the procaspase-3 variant containing the β-secretase cleavage sequence Gly-Ser-Ser-Glu-Ile-Ser-Tyr-Glu-Val-Glu-Phe-Arg-Glu-Phe (A). Activity was expressed in absorbance units (mean±SEM), and only β-secretase inhibitor IV elicited inhibition with an IC₅₀ of 19.8±2.4 nM. The three compounds were also tested against cathepsin B activity using the fluorogenic substrate Z-Arg-Arg AMC (mean fluorescence units±SEM plotted). β-secretase inhibitor IV had no effect on the cathepsin B activity, and PADK and CA074me resulted in IC₅₀ values of 9,200±1,030 and 120±13 nM, respectively (B).</p

PADK-modulated cathepsin B is localized to lysosomes.

<p>From PADK-treated mice (20 mg/kg/day×9 days) exhibiting increased levels of active cathepsin B, fixed hemi-brains were sectioned and double-stained for cathepsin B (green) and the lysosomal marker LAMP1 (red). Individual antigen labeling and the merged image from hippocampal CA1 show that PADK-modulated cathepsin B highly co-localizes with LAMP1-positive organelles in pyramidal neurons. View-field width: 35 µm. To localize cathepsin B activity, APP_SwInd mice were injected daily with vehicle (–) or 18 mg/kg PADK (+) for 10 days, and cortical and hippocampal regions were subsequently dissected to isolate lysosomes. Lysosomal fractions (Lys) and non-lysosomal fractions (non) were separated using Percoll gradients, and the two types of fractions were separately pooled and assessed for protein content and hydrolase activity with Z-Arg-Arg AMC (mean specific activity plotted±SEM). Unpaired Mann-Whitney U-test compared to lysosomal fractions from vehicle-treated mice: ***<i>P</i><0.0001.</p

PADK decreases 6E10 immunostaining in APP-PS1 mouse brain.

<p>APP-PS1 mice were injected i.p. daily with PADK (20 mg/kg; n = 11) or vehicle (n = 10) for 11 days. Fixed tissue was sectioned and stained with the 6E10 antibody. Image analysis for densitometric quantification of the immunostaining (mean integrated optical density±SEM) was conducted across view-fields of the hippocampal CA1 stratum pyramidale (sp). Area of deposit labeling above background was also measured for view-fields of the hippocampal stratum radiatum (sr) and piriform cortex (mean percent of total measured area±SEM). ANOVAs: <i>P</i><0.0001; Tukey's post hoc tests compared to APP−PS1+vehicle.</p><p>**<i>P</i><0.001.</p