Folding propensity of intrinsically disordered proteins by osmotic stress.

Proteins imparted with intrinsic disorder conduct a range of essential cellular functions. To better understand the folding and hydration properties of intrinsically disordered proteins (IDPs), we used osmotic stress to induce conformational changes in nuclear co-activator binding domain (NCBD) and activator for thyroid hormone and retinoid receptor (ACTR) separate from their mutual binding. Osmotic stress was applied by the addition of small and polymeric osmolytes, where we discovered that water contributions to NCBD folding always exceeded those for ACTR. Both NCBD and ACTR were found to gain α-helical structure with increasing osmotic stress, consistent with their folding upon NCBD/ACTR complex formation. Using small-angle neutron scattering (SANS), we further characterized NCBD structural changes with the osmolyte ethylene glycol. Here a large reduction in overall size initially occurred before substantial secondary structural change. By focusing on folding propensity, and linked hydration changes, we uncover new insights that may be important for how IDP folding contributes to binding.

Discovery of an inhibitor of Z-alpha1 antitrypsin polymerization.

Polymerization of the Z variant alpha-1-antitrypsin (Z-α1AT) results in the most common and severe form of α1AT deficiency (α1ATD), a debilitating genetic disorder whose clinical manifestations range from asymptomatic to fatal liver and/or lung disease. As the altered conformation of Z-α1AT and its attendant aggregation are responsible for pathogenesis, the polymerization process per se has become a major target for the development of therapeutics. Based on the ability of Z-α1AT to aggregate by recruiting the reactive center loop (RCL) of another Z-α1AT into its s4A cavity, we developed a high-throughput screening assay that uses a modified 6-mer peptide mimicking the RCL to screen for inhibitors of Z-α1AT polymer growth. A subset of compounds from the Library of Pharmacologically Active Compounds (LOPAC) with molecular weights ranging from 300 to 700 Da, was used to evaluate the assay's capabilities. The inhibitor S-(4-nitrobenzyl)-6-thioguanosine was identified as a lead compound and its ability to prevent Z-α1AT polymerization confirmed by secondary assays. To further investigate the binding location of S-(4-nitrobenzyl)-6-thioguanosine, an in silico strategy was pursued and the intermediate α1AT M* state modeled to allow molecular docking simulations and explore various potential binding sites. Docking results predict that S-(4-nitrobenzyl)-6-thioguanosine can bind at the s4A cavity and at the edge of ß-sheet A. The former binding site would directly block RCL insertion whereas the latter site would prevent ß-sheet A from expanding between s3A/s5A, and thus indirectly impede RCL insertion. Altogether, our investigations have revealed a novel compound that inhibits the formation of Z-α1AT polymers, as well as in vitro and in silico strategies for identifying and characterizing additional blocking molecules of Z-α1AT polymerization.

Investigating the structural impact of the glutamine repeat in huntingtin assembly.

Acquiring detailed structural information about the various aggregation states of the huntingtin-exon1 protein (Htt-exon1) is crucial not only for identifying the true nature of the neurotoxic species responsible for Huntington's disease (HD) but also for designing effective therapeutics. Using time-resolved small-angle neutron scattering (TR-SANS), we followed the conformational changes that occurred during fibrillization of the pathologic form of Htt-exon1 (NtQ42P10) and compared the results with those obtained for the wild-type (NtQ22P10). Our results show that the aggregation pathway of NtQ22P10 is very different from that of NtQ42P10, as the initial steps require a monomer to 7-mer transition stage. In contrast, the earliest species identified for NtQ42P10 are monomer and dimer. The divergent pathways ultimately result in NtQ22P10 fibrils that possess a packing arrangement consistent with the common amyloid sterical zipper model, whereas NtQ42P10 fibrils present a better fit to the Perutz ß-helix structural model. The structural details obtained by TR-SANS should help to delineate the key mechanisms that underpin Htt-exon1 aggregation leading to HD.

Structural formation of huntingtin exon 1 aggregates probed by small-angle neutron scattering.

In several neurodegenerative disorders, including Huntington's disease, aspects concerning the earliest of protein structures that form along the aggregation pathway have increasingly gained attention because these particular species are likely to be neurotoxic. We used time-resolved small-angle neutron scattering to probe in solution these transient structures formed by peptides having the N-terminal sequence context of mutant huntingtin exon 1. We obtained snapshots of the formed aggregates as the kinetic reaction ensued to yield quantitative information on their size and mass. At the early stage, small precursor species with an initial radius of gyration of 16.1 ± 5.9 Å and average mass of a dimer to trimer were monitored. Structural growth was treated as two modes with a transition from three-dimensional early aggregate formation to two-dimensional fibril growth and association. Our small-angle neutron scattering results on the internal structure of the mature fibrils demonstrate loose packing with ~1 peptide per 4.75 Åß-sheet repeat distance, which is shown to be quantitatively consistent with a ß-helix model. This research provides what we believe to be new insights into the structures forming along the pathway of huntingtin exon 1 aggregation and should assist in determining the role that precursors play in neuronal toxicity.

Screening for modulators of aggregation with a microplate elongation assay.

Many protein misfolding or conformational diseases, a number of which are neurodegenerative, are associated with the presence of proteinaceous deposits in the form of amyloid/amyloid-like fibrils/aggregates in tissues. Little is known about the exact mechanisms by which fibrillar aggregates are formed and can impair cellular functions leading to cell death. Small molecules that can modulate aggregate formation and/or structure can be powerful tools for studying the aggregate assembly mechanism and toxicity and may also prove to be therapeutic. We describe here a microplate-based high-throughput screening assay for identification of such molecules. The assay is based on the ability of microplate-coated aggregates to grow by incorporating additional monomers. Compounds that influence the elongation reaction are selected as hits and are tested in dose-response experiments. We also discuss some additional experiments that can be used to characterize the modes of action of these aggregation modulators further.

Imaging polyglutamine deposits in brain tissue.

The formation of polyglutamine aggregates occupies a central role in the pathophysiology of neurodegenerative diseases caused by expanded trinucleotide repeats encoding the amino acid glutamine. This chapter describes sensitive histological methods for detection of tissue sites that are capable of further recruitment of polyglutamine and for sites rich in polyglutamine defined immunohistochemically. These methods have been found to be applicable in a number of diseases and animal models of disease. Recruitment, which is a property of highly ordered, amyloid-like aggregates, is most commonly found in punctate sites, termed aggregation foci (AF), in the neuronal perikaryonal cytoplasm. As expected, these AF correspond to sites containing polyglutamine aggregates detected using the antibody 1C2. Interestingly, however, many of the latter sites, including most neuropil aggregates and neuronal intranuclear inclusions, exhibit a limited ability to support polyglutamine recruitment. Thus there is limited correlation between the distribution of polyglutamine aggregates and recruitment activity, suggesting functional heterogeneity among polyglutamine aggregates. These methods should prove useful in explaining the relationship between aggregation reactions, aggregate formation, and the development of symptomatic disease and should be adaptable to the study of other protein aggregation disorders.

Structural properties of Abeta protofibrils stabilized by a small molecule.

Metastable oligomeric and protofibrillar forms of amyloidogenic proteins have been implicated as on-pathway assembly intermediates in amyloid formation and as the major toxic species in a number of amyloid diseases including Alzheimer's disease. We describe here a chemical biology approach to structural analysis of Abeta protofibrils. Library screening yielded several molecules that stimulate Abeta aggregation. One of these compounds, calmidazolium chloride (CLC), rapidly and efficiently converts Abeta(1-40) monomers into clusters of protofibrils. As monitored by electron microscopy, these protofibrils persist for days when incubated in PBS at 37 degrees C, with a slow transition to fibrillar structures apparent only after several weeks. Like normal protofibrils, the CLC-Abeta aggregates exhibit a low thioflavin T response. Like Abeta fibrils, the clustered protofibrils bind the anti-amyloid Ab WO1. The CLC-Abeta aggregates exhibit the same protection from hydrogen-deuterium exchange as do protofibrils isolated from a spontaneous Abeta fibril formation reaction: approximately 12 of the 39 Abeta(1-40) backbone amide protons are protected from exchange in the protofibril, compared with approximately twice that number in amyloid fibrils. Scanning proline mutagenesis analysis shows that the Abeta molecule in these protofibrillar assemblies exhibits the same flexible N and C termini as do mature amyloid fibrils. The major difference in Abeta conformation between fibrils and protofibrils is added structural definition in the 22-29 segment in the fibril. Besides aiding structural analysis, compounds capable of facilitating oligomer and protofibril formation might have therapeutic potential, if they act to sequester Abeta in a form and/or location that cannot engage the toxic pathway.

Amyloid-like features of polyglutamine aggregates and their assembly kinetics.

The repeat length-dependent tendency of the polyglutamine sequences of certain proteins to form aggregates may underlie the cytotoxicity of these sequences in expanded CAG repeat diseases such as Huntington's disease. We report here a number of features of various polyglutamine (polyGln) aggregates and their assembly pathways that bear a resemblance to generally recognized defining features of amyloid fibrils. PolyGln aggregation kinetics displays concentration and length dependence and a lag phase that can be abbreviated by seeding. PolyGln aggregates exhibit classical beta-sheet-rich circular dichroism spectra consistent with an amyloid-like substructure. The fundamental structural unit of all the in vitro aggregates described here is a filament about 3 nm in width, resembling the protofibrillar intermediates in amyloid fibril assembly. We observed these filamentous structures either as isolated threads, as components of ribbonlike sheets, or, rarely, in amyloid-like twisted fibrils. All of the polyGln aggregates described here bind thioflavin T and shift its fluorescence spectrum. Although all polyGln aggregates tested bind the dye Congo red, only aggregates of a relatively long polyGln peptide exhibit Congo red birefringence, and this birefringence is only observed in a small portion of these aggregates. Remarkably, a monoclonal antibody with high selectivity for a generic amyloid fibril conformational epitope is capable of binding polyGln aggregates. Thus, polyGln aggregates exhibit most of the characteristic features of amyloid, but the twisted fibril structure with Congo red birefringence is not the predominant form in the polyGln repeat length range studied here. We also find that polyGln peptides exhibit an unusual freezing-dependent aggregation that appears to be caused by the freeze concentration of peptide and/or buffer components. This is of both fundamental and practical significance. PolyGln aggregation is revealed to be a highly specific process consistent with a significant degree of order in the molecular structure of the product. This ordered structure, or the assembly process leading to it, may be responsible for the cell-specific neuronal degeneration observed in Huntington's and other expanded CAG repeat diseases.