The Dynamic Interactions of a Multitargeting Domain in Ameloblastin Protein with Amelogenin and Membrane

The enamel matrix protein Ameloblastin (Ambn) has critical physiological functions, including regulation of mineral formation, cell differentiation, and cell–matrix adhesion. We investigated localized structural changes in Ambn during its interactions with its targets. We performed biophysical assays and used liposomes as a cell membrane model. The xAB2N and AB2 peptides were rationally designed to encompass regions of Ambn that contained self-assembly and helix-containing membrane-binding motifs. Electron paramagnetic resonance (EPR) on spin-labeled peptides showed localized structural gains in the presence of liposomes, amelogenin (Amel), and Ambn. Vesicle clearance and leakage assays indicated that peptide–membrane interactions were independent from peptide self-association. Tryptophan fluorescence and EPR showed competition between Ambn–Amel and Ambn–membrane interactions. We demonstrate localized structural changes in Ambn upon interaction with different targets via a multitargeting domain, spanning residues 57 to 90 of mouse Ambn. Structural changes of Ambn following its interaction with different targets have relevant implications for the multifunctionality of Ambn in enamel formation

Ameloblastin Binds to Phospholipid Bilayers via a Helix-Forming Motif within the Sequence Encoded by Exon 5

Ameloblastin (Ambn), the most abundant non-amelogenin enamel protein, is intrinsically disordered and has the potential to interact with other enamel proteins and with cell membranes. Here, through multiple biophysical methods, we investigated the interactions between Ambn and large unilamellar vesicles (LUVs), whose lipid compositions mimicked cell membranes involved in epithelial cell-extracellular matrix adhesion. Using a series of Ambn Trp/Phe variants and Ambn mutants, we further showed that Ambn binds to LUVs through a highly conserved motif within the sequence encoded by exon 5. Synthetic peptides derived from different regions of Ambn confirmed that the sequence encoded by exon 5 is involved in LUV binding. Sequence analysis of Ambn across different species showed that the N-terminus of this sequence contains a highly conserved motif with a propensity to form an amphipathic helix. Mutations in the helix-forming sequence resulted in a loss of peptide binding to LUVs. Our in vitro data suggest that Ambn binds the lipid membrane directly through a conserved helical motif and have implications for biological events such as Ambn-cell interactions, Ambn signaling, and Ambn secretion via secretory vesicles

Membrane Curvature-sensing and Curvature-inducing Activity of Islet Amyloid Polypeptide and Its Implications for Membrane Disruption.

Islet amyloid polypeptide (IAPP) is a 37-amino acid amyloid protein intimately associated with pancreatic islet β-cell dysfunction and death in type II diabetes. In this study, we combine spectroscopic methods and microscopy to investigate α-helical IAPP-membrane interactions. Using light scattering and fluorescence microscopy, we observe that larger vesicles become smaller upon treatment with human or rat IAPP. Electron microscopy shows the formation of various highly curved structures such as tubules or smaller vesicles in a membrane-remodeling process, and spectrofluorometric detection of vesicle leakage shows disruption of membrane integrity. This effect is stronger for human IAPP than for the less toxic rat IAPP. From CD spectra in the presence of different-sized vesicles, we also uncover the membrane curvature-sensing ability of IAPP and find that it transitions from inducing to sensing membrane curvature when lipid negative charge is decreased. Our in vivo EM images of immunogold-labeled rat IAPP and human IAPP show both forms to localize to mitochondrial cristae, which contain not only locally curved membranes but also phosphatidylethanolamine and cardiolipin, lipids with high spontaneous negative curvature. Disruption of membrane integrity by induction of membrane curvature could apply more broadly to other amyloid proteins and be responsible for membrane damage observed in other amyloid diseases as well

Membrane Curvature-sensing and Curvature-inducing Activity of Islet Amyloid Polypeptide and Its Implications for Membrane Disruption.

Islet amyloid polypeptide (IAPP) is a 37-amino acid amyloid protein intimately associated with pancreatic islet β-cell dysfunction and death in type II diabetes. In this study, we combine spectroscopic methods and microscopy to investigate α-helical IAPP-membrane interactions. Using light scattering and fluorescence microscopy, we observe that larger vesicles become smaller upon treatment with human or rat IAPP. Electron microscopy shows the formation of various highly curved structures such as tubules or smaller vesicles in a membrane-remodeling process, and spectrofluorometric detection of vesicle leakage shows disruption of membrane integrity. This effect is stronger for human IAPP than for the less toxic rat IAPP. From CD spectra in the presence of different-sized vesicles, we also uncover the membrane curvature-sensing ability of IAPP and find that it transitions from inducing to sensing membrane curvature when lipid negative charge is decreased. Our in vivo EM images of immunogold-labeled rat IAPP and human IAPP show both forms to localize to mitochondrial cristae, which contain not only locally curved membranes but also phosphatidylethanolamine and cardiolipin, lipids with high spontaneous negative curvature. Disruption of membrane integrity by induction of membrane curvature could apply more broadly to other amyloid proteins and be responsible for membrane damage observed in other amyloid diseases as well

ApoC-III Glycoforms Are Differentially Cleared by Hepatic TRL (Triglyceride-Rich Lipoprotein) Receptors.

ObjectiveApoC-III (apolipoprotein C-III) glycosylation can predict cardiovascular disease risk. Higher abundance of disialylated (apoC-III2) over monosialylated (apoC-III1) glycoforms is associated with lower plasma triglyceride levels. Yet, it remains unclear whether apoC-III glycosylation impacts TRL (triglyceride-rich lipoprotein) clearance and whether apoC-III antisense therapy (volanesorsen) affects distribution of apoC-III glycoforms. Approach and Results: To measure the abundance of human apoC-III glycoforms in plasma over time, human TRLs were injected into wild-type mice and mice lacking hepatic TRL clearance receptors, namely HSPGs (heparan sulfate proteoglycans) or both LDLR (low-density lipoprotein receptor) and LRP1 (LDLR-related protein 1). ApoC-III was more rapidly cleared in the absence of HSPG (t1/2=25.4 minutes) than in wild-type animals (t1/2=55.1 minutes). In contrast, deficiency of LDLR and LRP1 (t1/2=56.1 minutes) did not affect clearance of apoC-III. After injection, a significant increase in the relative abundance of apoC-III2 was observed in HSPG-deficient mice, whereas the opposite was observed in mice lacking LDLR and LRP1. In patients, abundance of plasma apoC-III glycoforms was assessed after placebo or volanesorsen administration. Volanesorsen treatment correlated with a statistically significant 1.4-fold increase in the relative abundance of apoC-III2 and a 15% decrease in that of apoC-III1. The decrease in relative apoC-III1 abundance was strongly correlated with decreased plasma triglyceride levels in patients.ConclusionsOur results indicate that HSPGs preferentially clear apoC-III2. In contrast, apoC-III1 is more effectively cleared by LDLR/LRP1. Clinically, the increase in the apoC-III2/apoC-III1 ratio on antisense lowering of apoC-III might reflect faster clearance of apoC-III1 because this metabolic shift associates with improved triglyceride levels

ApoC-III Glycoforms Are Differentially Cleared by Hepatic TRL (Triglyceride-Rich Lipoprotein) Receptors.

ObjectiveApoC-III (apolipoprotein C-III) glycosylation can predict cardiovascular disease risk. Higher abundance of disialylated (apoC-III2) over monosialylated (apoC-III1) glycoforms is associated with lower plasma triglyceride levels. Yet, it remains unclear whether apoC-III glycosylation impacts TRL (triglyceride-rich lipoprotein) clearance and whether apoC-III antisense therapy (volanesorsen) affects distribution of apoC-III glycoforms. Approach and Results: To measure the abundance of human apoC-III glycoforms in plasma over time, human TRLs were injected into wild-type mice and mice lacking hepatic TRL clearance receptors, namely HSPGs (heparan sulfate proteoglycans) or both LDLR (low-density lipoprotein receptor) and LRP1 (LDLR-related protein 1). ApoC-III was more rapidly cleared in the absence of HSPG (t1/2=25.4 minutes) than in wild-type animals (t1/2=55.1 minutes). In contrast, deficiency of LDLR and LRP1 (t1/2=56.1 minutes) did not affect clearance of apoC-III. After injection, a significant increase in the relative abundance of apoC-III2 was observed in HSPG-deficient mice, whereas the opposite was observed in mice lacking LDLR and LRP1. In patients, abundance of plasma apoC-III glycoforms was assessed after placebo or volanesorsen administration. Volanesorsen treatment correlated with a statistically significant 1.4-fold increase in the relative abundance of apoC-III2 and a 15% decrease in that of apoC-III1. The decrease in relative apoC-III1 abundance was strongly correlated with decreased plasma triglyceride levels in patients.ConclusionsOur results indicate that HSPGs preferentially clear apoC-III2. In contrast, apoC-III1 is more effectively cleared by LDLR/LRP1. Clinically, the increase in the apoC-III2/apoC-III1 ratio on antisense lowering of apoC-III might reflect faster clearance of apoC-III1 because this metabolic shift associates with improved triglyceride levels

Polyglutamine- and temperature-dependent conformational rigidity in mutant huntingtin revealed by immunoassays and circular dichroism spectroscopy.

BACKGROUND:In Huntington's disease, expansion of a CAG triplet repeat occurs in exon 1 of the huntingtin gene (HTT), resulting in a protein bearing>35 polyglutamine residues whose N-terminal fragments display a high propensity to misfold and aggregate. Recent data demonstrate that polyglutamine expansion results in conformational changes in the huntingtin protein (HTT), which likely influence its biological and biophysical properties. Developing assays to characterize and measure these conformational changes in isolated proteins and biological samples would advance the testing of novel therapeutic approaches aimed at correcting mutant HTT misfolding. Time-resolved Förster energy transfer (TR-FRET)-based assays represent high-throughput, homogeneous, sensitive immunoassays widely employed for the quantification of proteins of interest. TR-FRET is extremely sensitive to small distances and can therefore provide conformational information based on detection of exposure and relative position of epitopes present on the target protein as recognized by selective antibodies. We have previously reported TR-FRET assays to quantify HTT proteins based on the use of antibodies specific for different amino-terminal HTT epitopes. Here, we investigate the possibility of interrogating HTT protein conformation using these assays. METHODOLOGY/PRINCIPAL FINDINGS:By performing TR-FRET measurements on the same samples (purified recombinant proteins or lysates from cells expressing HTT fragments or full length protein) at different temperatures, we have discovered a temperature-dependent, reversible, polyglutamine-dependent conformational change of wild type and expanded mutant HTT proteins. Circular dichroism spectroscopy confirms the temperature and polyglutamine-dependent change in HTT structure, revealing an effect of polyglutamine length and of temperature on the alpha-helical content of the protein. CONCLUSIONS/SIGNIFICANCE:The temperature- and polyglutamine-dependent effects observed with TR-FRET on HTT proteins represent a simple, scalable, quantitative and sensitive assay to identify genetic and pharmacological modulators of mutant HTT conformation, and potentially to assess the relevance of conformational changes during onset and progression of Huntington's disease

The temperature dependence of the 2B7-MW1 TR-FRET signal is inversely proportional to polyQ length.

<p>A.-B. Fluorescence signals obtained from dilution curves of N548 proteins bearing Q16, Q19, Q25, Q33, Q39 and Q55, tested by TR-FRET (1 ng/µl of 2B7-Tb and 10 ng/µl of MW1-d2) at RT (A) and 4°C (B). C. Ratio between the maximum value of the TR-FRET signal obtained at 4°C and the maximum value of the TR-FRET signal obtained at RT, referred to the curves presented in A and B. D. Ratio obtained and expressed as in C after performing TR-FRET with 2B7-Tb (1 ng/µl) and MW1-d2 (10 ng/µl) antibody combination (as before) or MW1-Tb (1 ng/µl) and 2B7-Alexa647 (10 ng/µl) antibody combination on N548 proteins bearing Q16 and Q55 repeats. In C and D data are represented as mean ± S.D. of three independent experiments; significance was calculated using the one-way ANOVA test and Bonferroni's multiple comparison post-test (** p<0.01 and * p<0.05).</p

Temperature-dependent alpha-helical variations in thioredoxin-tagged exon 1 HTT proteins are not due to variations in tag helicity, and are also observed in untagged, larger N548 HTT purified recombinant proteins.

<p>A. MRE values at 222 nm (at which alpha-helical structure has a signature peak) for Q25 and Q46 THRX-HTT exon 1 at each temperature tested. Red asterisks denote measurements taken at 4°C and RT, the temperatures used in the TR-FRET experiments. Black asterisks denote <i>p</i>-values as determined by the Student's <i>t</i>-test. *<i>p</i><0.05. **<i>p</i><0.01. ns, not significant. All data are represented as mean ± S.D. of three independent experiments. B. CD spectra for the THRX fusion partner alone at indicated temperatures. C. MRE spectra for the Q25 N548 HTT protein at −10°C and 37°C, showing that the temperature effect can still be seen in this longer fragment. D. MRE values at 222 nm at each indicated temperature relative to MRE at 37°C for N548 HTT of increasing polyQ length (Q19, Q22, Q25, Q42, Q52, Q55).</p

Temperature- and polyQ- dependent conformational changes detected on HTT full length protein (Q18 and Q83) using the 2B7-MW1 TR-FRET assay in a biological sample (lysates from transfected cells).

<p>A–B. TR-FRET values obtained at RT (A) or 4°C (B) using 2B7-Tb (1 n/µl) - MW1-d2 (10 ng/µl) on lysates, confirming the temperature- and polyQ-dependent conformational effect. C. Absolute ratios between the maximum value of the TR-FRET signal obtained at 4°C and the maximum value of the TR-FRET signal obtained at RT, referred to the curves in A and B. D. Absolute ratios between the maximum value of the TR-FRET signal obtained at 4°C and the maximum value of the TR-FRET signal obtained at RT, referred to the curves achieved using a 1∶1 ratio of 2B7-Tb and MW1-d2 (1 n/µl for both antibodies). E-F. TR-FRET values obtained at RT (E) or 4°C (F) using 2B7-Tb and 4C9-Alexa647 on the same lysates, confirming the requirement for polyQ detection to observe conformational changes. In C, D and G data are represented as mean ± S.D. of three independent experiments; significance was calculated using the one-way ANOVA test and Bonferroni's multiple comparison post-test (** = p<0.01; *** = p<0.005).</p