Small molecule probes of protein aggregation

Understanding the mechanisms of amyloid formation and toxicity remain major challenges. Whilst substantial progress has been made in the development of methods able to identify the species formed during self-assembly and to describe the kinetic mechanisms of aggregation, the structure(s) of non-native species, including potentially toxic oligomers, remain elusive. Moreover, how fibrils contribute to disease remains unclear. Here we review recent advances in the development of small molecules and other reagents that are helping to define the mechanisms of protein aggregation in molecular detail. Such probes form a powerful platform with which to better define the mechanisms of structural conversion into amyloid fibrils and may provide the much-needed stepping stone for future development of successful therapeutic agents

Biological applications of designed hairpin peptides: as antimicrobials and as inhibitors of amyloidogenesis

Thesis (Ph.D.)--University of Washington, 2017-06More than 40 diseases have been associated with the misfolding of peptides (or proteins) that form fibrils with a very specific morphology. These peptides classified as amyloidogenic peptides have been implicated in the development of Alzheimer’s Disease, Parkinson’s Disease, Type II Diabetes, Hungtinton’s Disease etc. To date, these diseases have no cure, only therapies that can ameliorate the symptoms to a degree. Inhibition of the amyloidogenesis of these peptides has been proposed as a possible treatment option. While small molecules have been heavily tested as inhibitors of amyloidogenesis, peptides have emerged as potential inhibitors. In this work, the ability of a set of designed hairpin peptides to inhibit the amyloidogenesis of two different systems, α-synuclein (implicated in Parkinson’s Disease) and human amylin (implicated in Type II Diabetes) is tested. Using circular dichroism and thioflavin T fluorescence, the ability of these peptides to inhibit amyloidogenesis is tested. The binding loci of these inhibitors to α-synuclein are also explored. The use of peptides as antimicrobials on the other hand is not a novel concept. However, most antimicrobial peptides, both natural and designed, rely heavily on covalent stabilizations in order to maintain secondary structure. In this study, non-covalent stabilizations are applied to a couple of natural as well as designed antimicrobials in order to study the effects of secondary structure stabilization on biological activity

Inhibition of Human Amylin Amyloidogenesis by Human Amylin-Fragment Peptides: Exploring the Effects of Serine Residues and Oligomerization upon Inhibitory Potency

To date, fragments from within the amyloidogenic-patch region of human amylin (hAM) have been shown to aggregate independently of the full-length peptide. In this study, we show that under certain conditions, both oligomers of NFGAILSS and the monomeric form are capable of inhibiting the aggregation of the full-length hAM sequence. The inhibition, rather than aggregate seeding, observed with the soluble portion of aged NFGAILSS solutions was particularly striking occurring at far substoichiometric levels. Apparently, the oligomer form of this fragment is responsible for inhibiting the transition from random coil to β-sheet or serves as a disaggregator of hAM β-oligomers. Sequential deletion of the serine residues from NFGAILSS results in a decrease of inhibition, indicating that these residues are important to the activity of this fragment. We, like others, observed instances of α-helix-like CD spectra prior to β-sheet formation as part of the amyloidogenesis pathway. The partially aggregated sample and the fragments studied display spectroscopic diagnostics, suggesting that they slow down the conversion of full-length hAM monomers to cytotoxic oligomers

Binding Interactions of Agents That Alter \u3b1-Synuclein Aggregation.

Further examination of peptides with well-folded antiparallel beta strands as inhibitors of amyloid formation from alpha-synuclein has resulted in more potent inhibitors. Several of these had multiple Tyr residues and represent a new lead for inhibitor design by small peptides that do not divert alpha-synuclein to non-amyloid aggregate formation. The most potent inhibitor obtained in this study is a backbone cyclized version of a previously studied beta hairpin, designated as WW2, with a cross-strand Trp/Trp cluster. The cyclization was accomplished by adding a D-Pro-L-Pro turn locus across strand termini. At a 2 : 1 peptide to alpha-synuclein ratio, cyclo-WW2 displays complete inhibition of beta-structure formation. Trp-bearing antiparallel beta-sheets held together by a disulphide bond are also potent inhibitors. N-15 HSQC spectra of alpha-synuclein provided new mechanistic details. The time course of N-15 HSQC spectral changes observed during beta-oligomer formation has revealed which segments of the structure become part of the rigid core of an oligomer at early stages of amyloidogenesis and that the C-terminus remains fully flexible throughout the process. All of the effective peptide inhibitors display binding-associated titration shifts in N-15 HSQC spectra of alpha-synuclein in the C-terminal Q109-E137 segment. Cyclo-WW2, the most potent inhibitor, also displays titration shifts in the G41-T54 span of alpha-synuclein, an additional binding site. The earliest aggregation event appears to be centered about H50 which is also a binding site for our most potent inhibitor

Fluorescent Probes Reveal a Minimal Ligase Recognition Motif in the Prokaryotic Ubiquitin-like Protein from Mycobacterium tuberculosis

The prokaryotic ubiquitin-like protein (Pup)-based proteasomal system in the pathogen Mycobacterium tuberculosis (<i>Mtb</i>) is essential for its survival in a mammalian host. The Pup ligase enzyme, PafA, conjugates Pup to a suite of proteins targeted for proteasomal degradation and is necessary for persistent infection by <i>Mtb</i>. We report the design and application of fluorescent probes for use in elucidating the mechanisms of Pup and substrate recognition by PafA. Our studies revealed that the C-terminal 26 amino acid sequence of Pup is the minimal ligase recognition motif in <i>Mtb</i>. Specific hydrophobic residues within this sequence that are known to be important for the interactions of Pup with proteasomes are also critical for the activation of Pup by PafA