Pre-structured hydrophobic peptide ß-strands: A universal amyloid trap?

Amyloid fibril formation has long been studied because of the variety of proteins that are capable of adopting this structure despite sharing little sequence homology. This makes amyloid fibrils a challenging focus for inhibition studies because the peptides and proteins that form amyloid fibrils cannot be targeted based on a sequence motif. Most peptide inhibitors that target specific amyloidogenic proteins rely heavily on sequence recognition to ensure that the inhibitory peptide is able to bind its target. This approach is limited to targeting one amyloidogenic protein at a time. However, there is increasing evidence of cross-reactivity between amyloid-forming polypeptides. It has therefore become more useful to study the similarities between these proteins that goes beyond their sequence homology. Indeed, the observation that amyloidogenic proteins adopt similar secondary structures along the pathway to fibril formation opens the way to an interesting investigation: the development of inhibitors that could be universal amyloid traps. The review below will analyze two specific amyloidogenic proteins, α-synuclein and human amylin, and introduce a small number of peptides that have been shown to be capable of inhibiting the amyloidogenesis of both of these very dissimilar polypeptides. Some of the inhibitory peptide motifs may indeed, be applicable to Aß and other amyloidogenic systems.

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 ß-sheet or serves as a disaggregator of hAM ß-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 ß-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.

Biological consequences of improving the structural stability of hairpins that have antimicrobial activity.

Designing new antimicrobial peptides (AMPs) focuses heavily on the activity of the peptide and less on the elements that stabilize the secondary structure of these peptides. Studies have shown that improving the structure of naturally occurring AMPs can affect activity and so here we explore the relationship between structure and activity of two non-naturally occurring AMPs. We have used a backbone-cyclized peptide as a template and designed an uncyclized analogue of this peptide that has antimicrobial activity. We focused on beta-hairpin-like structuring features. Improvements to the structure of this peptide reduced the activity of the peptide against gram-negative, Escherichia coli but improved the activity against gram-positive, Corynebacterium glutamicum. Distinctions in structuring effects on gram-negative versus gram-positive activity were also seen in a second peptide system. Structural improvements resulted in a peptide that was more active than the native against gram-positive bacterium but less active against gram-negative bacterium. Our results show that there is not always a correlation between improved hairpin-structuring and activity. Other factors such as the type of bacteria being targeted as well as net positive charge can play a role in the potency of AMPs. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

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 (Mtb) 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 Mtb. 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 Mtb. 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.

MyD88-dependent signaling drives toll-like receptor-induced trained immunity in macrophages.

Immunocompromised populations are highly vulnerable to developing life-threatening infections. Strategies to protect patients with weak immune responses are urgently needed. Employing trained immunity, whereby innate leukocytes undergo reprogramming upon exposure to a microbial product and respond more robustly to subsequent infection, is a promising approach. Previously, we demonstrated that the TLR4 agonist monophosphoryl lipid A (MPLA) induces trained immunity and confers broad resistance to infection. TLR4 signals through both MyD88- and TRIF-dependent cascades, but the relative contribution of each pathway to induction of trained immunity is unknown. Here, we show that MPLA-induced resistance to Staphylococcus aureus infection is lost in MyD88-KO, but not TRIF-KO, mice. The MyD88-activating agonist CpG (TLR9 agonist), but not TRIF-activating Poly I:C (TLR3 agonist), protects against infection in a macrophage-dependent manner. MPLA- and CpG-induced augmentation of macrophage metabolism and antimicrobial functions is blunted in MyD88-, but not TRIF-KO, macrophages. Augmentation of antimicrobial functions occurs in parallel to metabolic reprogramming and is dependent, in part, on mTOR activation. Splenic macrophages from CpG-treated mice confirmed that TLR/MyD88-induced reprogramming occurs in vivo. TLR/MyD88-triggered metabolic and functional reprogramming was reproduced in human monocyte-derived macrophages. These data show that MyD88-dependent signaling is critical in TLR-mediated trained immunity.

Modulating the Amyloidogenesis of α-Synuclein.

Alpha-Synuclein is found in the neuronal cells but its native function is not well known. While α -synuclein is an intrinsically disordered protein that adopts a helical conformation upon membrane binding, numerous studies have shown that oligomeric ß-forms of this protein are cytotoxic. This response to misfolded species contributes to Parkinson's Disease etiology and symptoms. The resulting amyloid fibrils are an established diagnostic in Parkinson's Disease. In this review, we focus on strategies that have been used to inhibit the amyloidogenesis of α -synuclein either by stabilizing the native state, or by redirecting the pathway to less toxic aggregates. Small molecules such as polyphenols, peptides as well as large proteins have proven effective at protecting cells against the cytotoxicity of α-synuclein. These strategies may lead to the development of therapeutic agents that could prove useful in combating this disease.

Peptide Inhibitors of the amyloidogenesis of IAPP: verification of the hairpin-binding geometry hypothesis.

Versions of a previously discovered ß-hairpin peptide inhibitor of IAPP aggregation that are stabilized in that conformation, or even forced to remain in the hairpin conformation by a backbone cyclization constraint, display superior activity as inhibitors. The cyclized hairpin, cyclo-WW2, displays inhibitory activity at substoichiometric concentrations relative to this amyloidogenic peptide. The hairpin-binding hypothesis stands confirmed.

Hairpin structure stability plays a role in the activity of two antimicrobial peptides.

Many naturally occurring antimicrobial peptides (AMPs) are amphipathic with a ß-hairpin conformation stabilized by cross-strand disulfides across the associated ß-strands. Here, we show that the disulfides are not essential. Other structuring means such as better ß-turns and noncovalent cross-strand interactions can, with proper design, replace the disulfides with no loss in antimicrobial activity. Our results also demonstrate that the hairpin turn region may play a role in membrane recognition for at least one member of this class, since a homodimeric turnless ß-sheet analog showed no antimicrobial activity. We also examined the effects of N-terminal fatty acid adducts on AMPs. Surprisingly, the large hydrophobic carboxylic moieties examined completely eliminated the antimicrobial activity of previously active ß-hairpin peptides.