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Abstract: The assembly of proteins into large fibrillar aggregates, known as amyloid fibrils, is associated with a number of common and debilitating diseases. In some cases, proteins deposit extracellularly, while in others the aggregation is intracellular. A common feature of these diseases is the presence of aggregates of different sizes, including mature fibrils, small oligomeric precursors, and other less well understood structural forms such as amorphous aggregates. These various species possess distinct biochemical, biophysical, and pathological properties. Here, we detail a number of techniques that can be employed to examine amyloid fibrils and oligomers using a fluorescence-detection system (FDS) coupled with the analytical ultracentrifuge. Sedimentation velocity analysis using fluorescence detection is a particularly useful method for resolving the complex heterogeneity present in amyloid systems and can be used to characterize aggregation in exceptional detail. Furthermore, the fluorescence detection module provides a number of particularly attractive features for the analysis of aggregating proteins. It expands the practical range of concentrations of aggregating proteins under study, which is useful for greater insight into the aggregation process. It also enables the assessment of aggregation behavior in complex biological solutions, such as cell lysates, and the assessment of processes that regulate in-cell or extracellular aggregation kinetics. Four methods of fluorescent detection that are compatible with the current generation of FDS instrumentation are described: (1) Detection of soluble amyloid fibrils using a covalently bound fluorophore. (2) Detection of amyloid fibrils using an extrinsic dye that emits fluorescence when bound to fibrils. (3) Detection of fluorescently-labeled lipids and their interaction with oligomeric amyloid intermediates. (4) Detection of green fluorescence protein (GFP) constructs and their interactions within mammalian cell lysates. [Copyright &y& Elsevier]

Abstract: Plasma apolipoproteins show α-helical structure in the lipid-bound state and limited conformational stability in the absence of lipid. This structural instability of lipid-free apolipoproteins may account for the high propensity of apolipoproteins to aggregate and accumulate in disease-related amyloid deposits. Here, we explore the properties of amyloid fibrils formed by apolipoproteins using human apolipoprotein (apo) C-II as a model system. Hydrogen-deuterium exchange and NMR spectroscopy of apoC-II fibrils revealed core regions between residues 19–37 and 57–74 with reduced amide proton exchange rates compared to monomeric apoC-II. The C-terminal core region was also identified by partial proteolysis of apoC-II amyloid fibrils using endoproteinase GluC and proteinase K. Complete tryptic hydrolysis of apoC-II fibrils followed by centrifugation yielded a single peptide in the pellet fraction identified using mass spectrometry as apoC-II56-76. Synthetic apoC-II56-76 readily formed fibrils, albeit with a different morphology and thioflavinT fluorescence yield compared to full-length apoC-II. Studies with smaller peptides narrowed this fibril-forming core to a region within residues 60–70. We postulate that the ability of apoC-II60-70 to independently form amyloid fibrils drives fibril formation by apoC-II. These specific amyloid-forming regions within apolipoproteins may underlie the propensity of apolipoproteins and their peptide derivatives to accumulate in amyloid deposits in vivo. [Copyright &y& Elsevier]

Apolipoprotein-D is a 25 kDa glycosylated member of the lipocalin family that folds into an eight-stranded β-barrel with a single adjacent α-helix. Apolipoprotein-D specifically binds a range of small hydrophobic ligands such as progesterone and arachidonic acid and has an antioxidant function that is in part due to the reduction of peroxidised lipids by methionine-93. Therefore, apolipoprotein-D plays multiple roles throughout the body and is protective in Alzheimer’s disease, where apolipoprotein-D overexpression reduces the amyloid-β burden in Alzheimer’s disease mouse models. Oligomerisation is a common feature of lipocalins that can influence ligand binding. The native structure of apolipoprotein-D, however, has not been conclusively defined. Apolipoprotein-D is generally described as a monomeric protein, although it dimerises when reducing peroxidised lipids. Here, we investigated the native structure of apolipoprotein-D derived from plasma, breast cyst fluid (BCF) and cerebrospinal fluid. In plasma and cerebrospinal fluid, apolipoprotein-D was present in high-molecular weight complexes, potentially in association with lipoproteins. In contrast, apolipoprotein-D in BCF formed distinct oligomeric species. We assessed apolipoprotein-D oligomerisation using native apolipoprotein-D purified from BCF and a suite of complementary methods, including multi-angle laser light scattering, analytical ultracentrifugation and small-angle X-ray scattering. Our analyses showed that apolipoprotein-D predominantly forms a ∼95 to ∼100 kDa tetramer. Small-angle X-ray scattering analysis confirmed these findings and provided a structural model for apolipoprotein-D tetramer. These data indicate apolipoprotein-D rarely exists as a free monomer under physiological conditions and provide insights into novel native structures of apolipoprotein-D and into oligomerisation behaviour in the lipocalin family. [ABSTRACT FROM AUTHOR]

[Display omitted] • Small heat-shock proteins populate a monomer–dimer-oligomer equilibrium. • The alpha-crystallin domain binds at fibril ends to inhibit elongation. • Chaperone-activity is inhibited by disulfide crosslinking at the dimer interface. • Monomers of the alpha-crystallin domain are proposed to interact with fibril ends. Small heat-shock proteins (sHSPs) are ubiquitously expressed molecular chaperones present in all kingdoms of life that inhibit protein misfolding and aggregation. Despite their importance in proteostasis, the structure–function relationships of sHSPs remain elusive. Human sHSPs are characterised by a central, highly conserved α-crystallin domain (ACD) and variable-length N- and C-terminal regions. The ACD forms antiparallel homodimers via an extended β-strand, creating a shared β-sheet at the dimer interface. The N- and C-terminal regions mediate formation of higher order oligomers that are thought to act as storage forms for chaperone-active dimers. We investigated the interactions of the ACD of two human sHSPs, αB-crystallin (αB-C) and Hsp27, with apolipoprotein C-II amyloid fibrils using analytical ultracentrifugation and nuclear magnetic resonance spectroscopy. The ACD was found to interact transiently with amyloid fibrils to inhibit fibril elongation and naturally occurring fibril end-to-end joining. This interaction was sensitive to the concentration of fibril ends indicating a 'fibril-capping' interaction. Furthermore, resonances arising from the ACD monomer were attenuated to a greater extent than those of the ACD dimer in the presence of fibrils, suggesting that the monomer may bind fibrils. This hypothesis was supported by mutagenesis studies in which disulfide cross-linked ACD dimers formed by both αB-C and Hsp27 were less effective at inhibiting amyloid fibril elongation and fibril end-to-end joining than ACD constructs lacking disulfide cross-linking. Our results indicate that sHSP monomers inhibit amyloid fibril elongation, highlighting the importance of the dynamic oligomeric nature of sHSPs for client binding. [ABSTRACT FROM AUTHOR]

Abstract: The misfolding and aggregation of proteins to form amyloid fibrils is a characteristic feature of several common age-related diseases. Agents that directly inhibit formation of amyloid fibrils represent one approach to combating these diseases. We have investigated the potential of a cyclic peptide to inhibit fibril formation by fibrillogenic peptides from human apolipoprotein C-II (apoC-II). Cyc[60–70] was formed by disulfide cross-linking of cysteine residues added to the termini of the fibrillogenic peptide comprising apoC-II residues 60–70. This cyclic peptide did not self-associate into fibrils. However, substoichiometric concentrations of cyc[60–70] significantly delayed fibril formation by the fibrillogenic, linear peptides apoC-II[60–70] and apoC-II[56–76]. Reduction of the disulfide bond or scrambling the amino acid sequence within cyc[60–70] significantly impaired its inhibitory activity. The solution structure of cyc[60–70] was solved using NMR spectroscopy, revealing a well-defined structure comprising a hydrophilic face and a more hydrophobic face containing the Met60, Tyr63, Ile66 and Phe67 side chains. Molecular dynamics (MD) studies identified a flexible central region within cyc[60–70], while MD simulations of “scrambled” cyc[60–70] indicated an increased formation of intramolecular hydrogen bonds and a reduction in the overall flexibility of the peptide. Our structural studies suggest that the inhibitory activity of cyc[60–70] is mediated by an elongated structure with inherent flexibility and distinct hydrophobic and hydrophilic faces, enabling cyc[60–70] to interact transiently with fibrillogenic peptides and inhibit fibril assembly. These results suggest that cyclic peptides based on amyloidogenic core peptides could be useful as specific inhibitors of amyloid fibril formation. [Copyright &y& Elsevier]

Abstract: GSTD1 is one of several insect glutathione S-transferases capable of metabolizing the insecticide DDT. Here we use crystallography and NMR to elucidate the binding of DDT and glutathione to GSTD1. The crystal structure of Drosophila melanogaster GSTD1 has been determined to 1.1 Å resolution, which reveals that the enzyme adopts the canonical GST fold but with a partially occluded active site caused by the packing of a C-terminal helix against one wall of the binding site for substrates. This helix would need to unwind or be displaced to enable catalysis. When the C-terminal helix is removed from the model of the crystal structure, DDT can be computationally docked into the active site in an orientation favoring catalysis. Two-dimensional 1H,15N heteronuclear single-quantum coherence NMR experiments of GSTD1 indicate that conformational changes occur upon glutathione and DDT binding and the residues that broaden upon DDT binding support the predicted binding site. We also show that the ancestral GSTD1 is likely to have possessed DDT dehydrochlorinase activity because both GSTD1 from D. melanogaster and its sibling species, Drosophila simulans, have this activity. [Copyright &y& Elsevier]

[Display omitted] • H2 relaxin has distinct and unique binding sites for RXFP1 and RXFP2. • Both the LDLa module and linker of RXFP1 and RXFP2 are essential for activation. • A conserved motif in the linker adopts different structures in RXFP1 and RXFP2. • These differences lead to distinct mechanisms for relaxin-induced activation. Our poor understanding of the mechanism by which the peptide-hormone H2 relaxin activates its G protein coupled receptor, RXFP1 and the related receptor RXFP2, has hindered progress in its therapeutic development. Both receptors possess large ectodomains, which bind H2 relaxin, and contain an N-terminal LDLa module that is essential for receptor signaling and postulated to be a tethered agonist. Here, we show that a conserved motif (GDxxGWxxxF), C-terminal to the LDLa module, is critical for receptor activity. Importantly, this motif adopts different structures in RXFP1 and RXFP2, suggesting distinct activation mechanisms. For RXFP1, the motif is flexible, weakly associates with the LDLa module, and requires H2 relaxin binding to stabilize an active conformation. Conversely, the GDxxGWxxxF motif in RXFP2 is more closely associated with the LDLa module, forming an essential binding interface for H2 relaxin. These differences in the activation mechanism will aid drug development targeting these receptors. [ABSTRACT FROM AUTHOR]