Structural Characterization of the Caveolin Scaffolding Domain in Association with Cholesterol-Rich Membranes

Members of the caveolin protein family are implicated in the formation of caveolae and play important roles in a number of signaling pathways and in the regulation of various proteins. We employ complementary spectroscopic methods to study the structure of the caveolin scaffolding domain (CSD) in caveolin-1 fragments, while bound to cholesterol-rich membranes. This key domain is thought to be involved in multiple critical functions that include protein recognition, oligomerization, and cholesterol binding. In our membrane-bound peptides, residues within the flanking intramembrane domain (IMD) are found to adopt an α-helical structure, consistent with its commonly believed helical hairpin conformation. Intriguingly, in these same peptides, we observe a β-stranded conformation for residues in the CSD, contrasting with earlier reports, which commonly do not reflect β-structure. Our experimental data based on solid-state NMR, CD, and FTIR are found to be consistent with computational analyses of the secondary structure preference of the primary sequence. We discuss how our structural data of membrane binding Cav fragments may match certain general features of cholesterol-binding domains and could be consistent with the role for CSD in protein recognition and homo-oligomerization

Aggregation time course of HTT^NTQ₃₇P₁₀C*K₂ by EM and FCS.

<p>Aggregation time course of HTT^NTQ₃₇P₁₀C*K₂ by EM and FCS.</p

Self-assembly of full length HTT exon1-EGFP fusions in PC12 living cells and cell extracts.

<p>A. Raw FCS data from clarified native lysates of PC12 cells after different growth times. B. Autocorrelation functions with residuals of 24 hrs clarified native lysates of cells producing GFP alone (green) or HTT exon1-Q₂₅-EGFP (black). C. Autocorrelation functions with residuals of native lysate supernatants of PC12 cells producing HTT exon1-Q₉₇-EGFP at different growth times (color code as in part A). D, E. Autocorrelation functions with residuals of data collected from the cytoplasm of living PC12 cells producing either HTT exon1-Q₂₅-EGFP (D) or HTT exon1-Q₉₇-EGFP (E).</p

Summary of FCS data showing molecular sizes under various conditions.

<p>Summary of FCS data showing molecular sizes under various conditions.</p

Structures and assembly mechanism of HTT exon1 polypeptides.

<p>A. Previously proposed mechanism of amyloid nucleation (HTT^NT, green; polyQ, orange; PRD, black). B. Sequence of HTT exon1. C. Sequences of peptides studied. C* = Cys residue modified with Alexa Fluor 555 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155747#sec009" target="_blank">Materials and Methods</a>).</p

Self-assembly in PBS of chemically synthesized HTT exon1 analogs.

<p>A. Raw FCS time-dependent fluorescence fluctuations of HTT^NTQ₂₃P₁₀C*K₂ (red) and HTT^NTQ₃₇P₁₀C*K₂ (black). B. Autocorrelation functions for the 10 mins FCS data shown in A, with the data points as filled squares and solid lines representing the fits (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155747#sec009" target="_blank">Materials and Methods</a>) and the same color scheme as A. Lines below the graph show the residuals between the data points and the fit curve. C. Concentration dependence of molecular size estimated from diffusion times for HTT^NTQ₃₇P₁₀C*K₂ (■) and HTT^NTQ₂₃P₁₀C*K₂ (). D. EM detail of different time points from the PBS incubation of a mixture of 2.0 μM HTT^NTQ₃₇P₁₀K₂. (More EM data is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155747#pone.0155747.s001" target="_blank">S1 Fig</a>.)</p

Characteristics of the PC12 model.

<p>A. Confocal microscopy of the Q25 and Q97 versions of HTT exon1 after 6 and 24 hrs growth in the presence of 1 μM ponesterone. Blue = Hoechst dye stained nucleus; green = EGFP. B. Number of cells containing inclusions at different growth times determined by fluorescence microscopy. C. Percent cell death at different growth times as determined by the amount of the intracellular enzyme lactate dehydrogenase released into the medium, an indication of the loss of outer cell membrane integrity.</p

Kinetically Competing Huntingtin Aggregation Pathways Control Amyloid Polymorphism and Properties

In polyglutamine (polyQ) containing fragments of the Huntington’s disease protein huntingtin (htt), the N-terminal 17 amino acid htt^NT segment serves as the core of α-helical oligomers whose reversible assembly locally concentrates the polyQ segments, thereby facilitating polyQ amyloid nucleation. A variety of aggregation inhibitors have been described that achieve their effects by neutralizing this concentrating function of the htt^NT segment. In this paper we characterize the nature and limits of this inhibition for three means of suppressing htt^NT-mediated aggregation. We show that the previously described action of htt^NT peptide-based inhibitors is solely due to their ability to suppress the htt^NT-mediated aggregation pathway. That is, under htt^NT inhibition, nucleation of polyQ amyloid formation by a previously described alternative nucleation mechanism proceeds unabated and transiently dominates the aggregation process. Removal of the bulk of the htt^NT segment by proteolysis or mutagenesis also blocks the htt^NT-mediated pathway, allowing the alternative nucleation pathway to dominate. In contrast, the previously described immunoglobulin-based inhibitor, the antihtt^NT V_L 12.3 protein, effectively blocks both amyloid pathways, leading to stable accumulation of nonamyloid oligomers. These data show that the htt^NT-dependent and -independent pathways of amyloid nucleation in polyQ-containing htt fragments are in direct kinetic competition. The results illustrate how amyloid polymorphism depends on assembly mechanism and kinetics and have implications for how the intracellular environment can influence aggregation pathways