UCLA

I began my research in the Eisenberg laboratory by studying the polymorphic nature of amyloid proteins Islet Amyloid Polypeptide (IAPP), a protein whose plaques are implicated in Type II Diabetes, and Amyloid-beta (Abeta), whose aggregates were implicated in Alzheimer's Disease. Both polypeptides are cleavage products of precursor proteins, are intrinsically disordered, and contain a highly amyloidogenic C-terminus. Later, I expanded the study of polymorphism to tumor suppressor protein p53, whose aggregation has recently been associated with tumor progression. The last part of my dissertation involves the studies on what may constitute the toxic species of amyloid, involving work with segments from alpha-B-crystallin, IAPP, and paralogs of 53.

This dissertation begins with work on structural and kinetic characterization of IAPP using transmission electron microscopy (TEM) and thioflavin T dye-binding assays. Here, I worked under Jed Wiltzius, who had solved several crystal structures of segments of the polypeptide each of which formed in-register steric zippers. I aided in his studies by performing EM on several of the segments and confirmed they indeed formed fibrils in vitro. I also performed EM and kinetic assays on full-length mutant and wild-type human IAPP, providing evidence that IAPP is capable of forming two distinct fibril polymorphs originating from two different steric zipper spines. These results that illustrate the molecular basis for fibril polymorphism of IAPP suggests a mechanism of protein-only encoded information transfer of different prion strains.

To further understand the polymorphic nature of amyloid proteins, I then focused on structural characterization of Abeta. To elucidate Abeta polymorphism in atomic detail, my colleagues Jacques-Philippe Colletier, Arthur Laganowsky, Meytal Landau and I determined eight new micro-crystal structures of fibril-forming segments of Abeta. These structures, all of various forms of steric zippers, reveal a variety of modes of self-association of Abeta. Combining these atomic structures with previous nuclear magnetic resonance and electron tomography studies, we propose several fiber models, offering molecular models that further illustrate the polydispersity of Abeta assemblies. These structures and molecular models contribute fundamental information for understanding Abeta polymorphic nature and pathogenesis. We furthermore suggest that steric zipper interactions are also the core of protafilaments binding together, explaining the immense heterogeneity in fibril morphologies as visualized under EM and various other characterization methods.

Structural characterization of fibril formation was carried to a third protein, tumor suppressor p53. It had recently been suggested that amyloid aggregation of mutant p53 may account for its gain of toxic function in cancer cells. Working with Alice Soragni, we elucidated the atomic details of the spine of p53 fibrils by identifying the aggregation-prone region and crystallizing two overlapping segments within the region. I also characterized a third segment that appears to exhibit a different type of steric zipper packing than other two segments. Results show that this short region within p53 displays the amyloid fibril polymorphism exhibited by Abeta and IAPP. In addition, these structures provide the basis for structure-based design of inhibitors of p53 aggregation as a potential cancer therapeutic.

A recent structure of a toxic amyloid oligomer, termed cylindrin, led me to also focus on a preliminary analysis of the mechanism of toxicity of this segment from alpha-B-crystallin. This was work done in collaboration with Arthur Laganowsky. I performed liposome disruption assays on the peptide, which suggests that the mechanism of toxicity of cylindrin may not be through membrane disruption. In addition, in collaboration with Professor Alex Van der Bliek, I attempted to transgenically express the peptide in C. elegans, as an in vivo model to examine toxicity. It appears cylindrin expression in C. elegans may induce slight toxicity, as it induces autophagosome accumulation and a slightly longer lifespan and larger brood size in the worms.

Finally, motivated by the extreme difficulty in crystallizing segments of amyloid proteins longer than eight residues, I helped in developing a methodology that has the potential to improve the chances of crystallizing proteins whose structure has remained elusive. In collaboration with Arthur Laganowsky, Minglei Zhao and Professor Todd Yeates, we developed a new crystallization approach, termed metal-mediated synthetic symmetrization, that introduces pairs of histidine or cysteine mutations onto the surface of target proteins, and, upon coordination with metal, generates novel crystal lattice contacts or oligomeric assemblies, thus producing a variety of new crystal forms, and increasing the chances of growing diffraction-quality crystals. We examined the method on two model fusion proteins, T4 lysozyme (T4L) and maltose-binding protein (MBP), and the approach resulted in 16 new crystal structures displaying a variety of oligomeric assemblies and packing modes, representing new and distinct crystal forms for these proteins. The results suggest this method has potential utlility for crystallizing target proteins of unknown structure through either direct mutations on the target protein or fusion of the target protein to metal-site mutants of T4L or MBP, which could serve as crystallization chaperones.

Current work involves exploring non-typical steric zipper interactions. I have recently solved 3 more crystal structures of various segments of IAPP, one of which forms an out-of-register steric zipper. I have also solved 2 more out-of-register zipper structures of segments within p63 and p73, both paralogs of p53 and suggested to co-aggregate with mutant p53. Analysis of these out-of-register structures show that there is no weak interface among the hydrogen bonding interactions, unlike other structures that have displayed out-of-register packing. Interestingly, cell viability assays showed that these peptides are not very toxic, suggesting the importance of these weak interfaces in amyloid toxicity. This work further confirms the polymorphic nature of amyloids.

The results embodied in this dissertation have assisted in advancing our understanding of molecular basis for amyloid fibril polymorphism and provides a preliminary characterization of the potential toxic amyloid oligomer cylindrin. In addition, the new crystallization methodology described in this work has the potential to improve the chances of crystallizing longer amyloid segments and additional proteins of unknown structure. Greater comprehension of the structural details of amyloid proteins not only can shed light into amyloid-aggregation mechanisms, but can also offer insight into the mechanisms of toxicity and aid in the development of therapeutics that target amyloid fibrillization and block aggregation.