Selecting Multitarget Peptides for Alzheimer's Disease.

Alzheimer's disease (AD) is a multifactorial disease with a complex pathogenesis. Developing multitarget drugs could be a powerful strategy to impact the progressive loss of cognitive functions in this disease. The purpose of this study is to select a multitarget lead peptide candidate among a series of peptide variants derived from the neutrophil granule protein cathepsin G. We screened eight peptide candidates using the following criteria: (1) Inhibition and reversion of amyloid beta (Aß) oligomers, quantified using an enzyme-linked immunosorbent assay (ELISA); (2) direct binding of peptide candidates to the human receptor for advanced glycation end-products (RAGE), the Toll-like receptor 4 (TLR4) and the S100 calcium-binding protein A9 (S100A9), quantified by ELISA; (3) protection against Aß oligomer-induced neuronal cell death, using trypan blue to measure cell death in a murine neuronal cell line; (4) inhibition of TLR4 activation by S100A9, using a human TLR4 reporter cell line. We selected a 27-mer lead peptide that fulfilled these four criteria. This lead peptide is a privileged structure that displays inherent multitarget activity. This peptide is expected to significantly impact cognitive decline in mouse models of Alzheimer's disease, by targeting both neuroinflammation and neurodegeneration.

Neutrophil Granule Proteins Inhibit Amyloid Beta Aggregation and Neurotoxicity.

BACKGROUND: A role for neutrophils in the pathogenesis of Alzheimer's disease (AD) is emerging. We previously showed that the neutrophil granule proteins cationic antimicrobial protein of 37 kDa (CAP37), cathepsin G (CG), and neutrophil elastase (NE) directly bind the amyloid-beta peptide Aß1-42, a central player in AD pathogenesis. CAP37, CG, and NE are serine proteases that can cleave Aß1-42 at different sites and with different catalytic activities. OBJECTIVE: In this study, we compared the effects of these three proteins on Aß1-42 fibrillation and neurotoxicity. METHODS: Using mass spectrometry and in vitro aggregation assay, we found that NE and CG efficiently cleave Aß1-42. This cleavage correlates well with the inhibition of Aß1-42 aggregation into fibrils. In contrast, CAP37 did not efficiently cleave Aß1-42, but was still able to inhibit its fibrillation, most likely through a quenching effect. Inhibition of Aß1-42 aggregation by NE and CG neutralized its toxicity measured in cultured neurons. In contrast, inhibition of Aß1-42 aggregation by CAP37 did not inhibit its neurotoxicity. RESULTS: We found that a peptide derived from CAP37 could mimic the quenching and inhibition of Aß1-42 aggregation effects of the full-length protein. Additionally, this peptide was able to inhibit the neurotoxicity of the most toxic Aß1-42 aggregate, an effect that was not found with the full-length CAP37. CONCLUSION: These results shed light on the mechanisms of action of neutrophil granule proteins with regard to inhibition of Aß1-42 aggregation and neurotoxicity and open up a possible strategy for the discovery of new disease-modifying drugs for AD.

Antimicrobial Peptides Derived from the Immune Defense Protein CAP37 Inhibit TLR4 Activation by S100A9.

Purpose: Corneal abrasion is a common eye injury, and its resolution can be seriously complicated by bacterial infection. We showed that topical application of the cationic antimicrobial protein of 37 kDa (CAP37) promotes corneal re-epithelialization in mice, and peptides derived from CAP37 can recapitulate the antibacterial and wound-healing effects of the full-length protein. The current study was designed to identify the molecular mechanisms mediating the wound-healing effect of CAP37 and derived bioactive peptides. Methods: We used a TriCEPS-based, ligand-receptor glycocapture method to identify the binding partners of CAP37 on live human corneal epithelial cells using the hTCEpi cell line. We used an ELISA method to confirm binding with identified partners and test the binding with CAP37-derived peptides. We used a reporter cell line to measure activation of the identified membrane receptor by CAP37 and derived peptides. Results: We pulled down S100 calcium-binding protein A9 (S100A9) as a binding partner of CAP37 and found that CAP37 and four derived peptides encompassing two regions of CAP37 bind S100A9 with high affinities. We found that CAP37 and the S100A9-binding peptides could also directly interact with the Toll-like receptor 4 (TLR4), a known receptor for S100A9. CAP37 and one peptide partially activated TLR4. The other three peptides did not activate TLR4. Finally, we found that CAP37 and all four peptides could inhibit the activation of TLR4 by S100A9. Conclusions: This study identifies a mechanism of action for CAP37 and derived antimicrobial peptides that may restrain inflammatory responses to corneal injury and favor corneal re-epithelialization.

Self-regenerating giant hyaluronan polymer brushes.

Tailoring interfaces with polymer brushes is a commonly used strategy to create functional materials for numerous applications. Existing methods are limited in brush thickness, the ability to generate high-density brushes of biopolymers, and the potential for regeneration. Here we introduce a scheme to synthesize ultra-thick regenerating hyaluronan polymer brushes using hyaluronan synthase. The platform provides a dynamic interface with tunable brush heights that extend up to 20 microns - two orders of magnitude thicker than standard brushes. The brushes are easily sculpted into micropatterned landscapes by photo-deactivation of the enzyme. Further, they provide a continuous source of megadalton hyaluronan or they can be covalently-stabilized to the surface. Stabilized brushes exhibit superb resistance to biofilms, yet are locally digested by fibroblasts. This brush technology provides opportunities in a range of arenas including regenerating tailorable biointerfaces for implants, wound healing or lubrication as well as fundamental studies of the glycocalyx and polymer physics.

Hyaluronan synthase assembles hyaluronan on a [GlcNAc(ß1,4)]n-GlcNAc(α1→)UDP primer and hyaluronan retains this residual chitin oligomer as a cap at the nonreducing end.

Class I hyaluronan synthases (HAS) assemble [GlcNAc(ß1,4)GlcUA(ß1,3)]n-UDP at the reducing end and also make chitin. Streptococcus equisimilis HAS (SeHAS) also synthesizes chitin-UDP oligosaccharides, (GlcNAc-ß1,4)n-GlcNAc(α1→)UDP (Weigel et al. 2015). Here we determined if HAS uses chitin-UDPs as primers to initiate HA synthesis, leaving the non-HA primer at the nonreducing (NR) end. HA made by SeHAS membranes was purified, digested with streptomyces lyase, and hydrophobic oligomers were enriched by solid phase extraction and analyzed by MALDI-TOF MS. Jack bean hexosaminidase (JBH) and MS/MS were used to analyze 19 m/z species of possible GnHn ions with clustered GlcNAc (G) residues attached to disaccharide units (H): (GlcNAcß1,4)2-5[GlcUA(ß1,3)GlcNAc]2-6. JBH digestion sequentially removed GlcNAc from the NR-end of GnHn oligomers, producing successively smaller GnH2-3 series members. Since lyase releases dehydro-oligos (dHn; M-18), only the unique NR-end oligo lacks dehydro-GlcUA. Hn oligomers were undetectable in lyase digests, whereas JBH treatment created new H2-6m/z peaks (i.e. HA tetra- through dodeca-oligomers). MS/MS of larger GnHn species produced chitin (2-5 GlcNAcs), HA oligomers and multiple smaller series members with fewer GlcNAcs. All NR-ends (97%) started with GlcNAc, as a chitin trimer (three GlcNAcs), indicating that GlcNAc(ß1,4)2GlcNAc(α1→)-UDP may be optimal for initiation of HA synthesis. Also, HA made by live S. pyogenes cells had G4Hn chitin-oligo NR-ends. We conclude that chitin-UDP functions in vitro and in live cells as a primer to initiate synthesis of all HA chains and these primers remain at the NR-ends of HA chains as residual chitin caps [(GlcNAc-ß1,4)3-4].

Hyaluronan synthase control of synthesis rate and hyaluronan product size are independent functions differentially affected by mutations in a conserved tandem B-X7-B motif.

Hyaluronan synthases (HAS) normally make large (>MDa) hyaluronan (HA) products. Smaller HA fragments (e.g. 100-400 kDa) produced in vivo are associated with inflammation and cell signaling by HA receptors that bind small, but not large, HA. Although HA fragments can arise from breakdown by hyaluronidases, HAS might also be regulated directly to synthesize small HA. Here we examined the Streptococcus equisimilis HAS (SeHAS) C-terminus, which contains a tandem B-X7-B motif (K398-X7-R406-X7-K414), by testing the effects of 27 site-specific scanning mutations and 7 C-terminal truncations on HA synthesis activity and weight-average mass. Although HAS enzymes cannot be HA-binding proteins, these motifs are highly conserved within the Class I HAS family. Fifteen Arg406 mutants made large MDa HA (86-110% wildtype size), with specific activities from 70% to 177% of wildtype. In contrast, 10 of 12 Lys398 mutants made HA that was 8-14% of wildtype size (≤250-480 kDa), with specific activities from 14% to 64% of wildtype. Four nearly inactive (2% wildtype activity) C-terminal truncation mutants made MDa HA (56-71% wildtype). The results confirm earlier findings with Cys-mutants [Weigel PH, Baggenstoss BA. 2012. Hyaluronan synthase polymerizing activity and control of product size are discrete enzyme functions that can be uncoupled by mutagenesis of conserved cysteines. Glycobiology 22:1302-1310] that HAS uses two independent activities to control HA size and HA synthesis rate; these are two separate functions. We conclude that HAS regulatory modifications that alter tandem B-X7-B motif conformation could mimic these mutagenesis-induced effects, allowing HAS in vivo to make small HA directly. The results also support a model in which the tandem-motif region is part of the intra-HAS pore and interacts directly with HA.

Hyaluronan synthase assembles chitin oligomers with -GlcNAc(α1→)UDP at the reducing end.

Class I hyaluronan synthases (HASs) assemble a polysaccharide containing the repeating disaccharide [GlcNAc(ß1,4)GlcUA(ß1,3)]n-UDP and vertebrate HASs also assemble (GlcNAc-ß1,4)n homo-oligomers (chitin) in the absence of GlcUA-UDP. This multi-membrane domain CAZy GT2 family glycosyltransferase, which couples HA synthesis and translocation across the cell membrane, is atypical in that monosaccharides are incrementally assembled at the reducing, rather than the non-reducing, end of the growing polymer. Using Escherichia coli membranes containing recombinant Streptococcus equisimilis HAS, we demonstrate that a prokaryotic Class I HAS also synthesizes chitin oligomers (up to 15-mers, based on MS and MS/MS analyses of permethylated products). Furthermore, chitin oligomers were found attached at their reducing end to -4GlcNAc(α1→)UDP [i.e. (GlcNAcß1,4)nGlcNAc(α1→)UDP]. These oligomers, which contained up to at least seven HexNAc residues, consisted of ß4-linked GlcNAc residues, based on the sensitivity of the native products to jack bean ß-N-acetylhexosaminidase. Interestingly, these oligomers exhibited mass defects of -2, or -4 for longer oligomers, that strictly depended on conjugation to UDP, but MS/MS analyses indicate that these species result from chemical dehydrogenations occurring in the gas phase. Identification of (GlcNAc-ß1,4)n-GlcNAc(α1→)UDP as HAS reaction products, made in the presence of GlcNAc(α1→)UDP only, provides strong independent confirmation for the reducing terminal addition mechanism. We conclude that chitin oligomer products made by HAS are derived from the cleavage of these novel activated oligo-chitosyl-UDP oligomers. Furthermore, it is possible that these UDP-activated chitin oligomers could serve as self-assembled primers for initiating HA synthesis and ultimately modify the non-reducing terminus of HA with a chitin cap.