Self-assembled peptides: from nanostructure to bioactivity

In autoimmune diseases, it has been proposed that exogenous "molecular triggers", i.e., specific non-self-antigens accompanying infectious agents, may disrupt the control of adaptive immune system resulting in serious pathologies. The aetiology of multiple sclerosis (MS) remains unclear. However, epidemiologic data suggest that exposure to infectious agents may be associated with increased MS risk and that progression may be linked to exogenous, bacterially-derived, antigenic molecules mimicking mammalian cell surface glycoconjugates triggering autoimmune responses. Previously, antibodies specific to a gluco-asparagine (N-Glc) glycopeptide, CSF114(N-Glc), were identified in sera of an MS patient subpopulation. Since the human glycoproteome repertoire lacks this uniquely modified amino acid, the group turned its attention to bacteria, i.e., Haemophilus influenzae, expressing cell-surface adhesins including N-Glc, to establish connection between H. influenzae infection and MS. The group exploited the biosynthetic machinery from H. influenzae opportunistic pathogens (and the homologous enzymes from A. pleuropneumoniae) to produce a unique set of defined glucosylated adhesin proteins. Interestingly it revealed that a hyperglucosylated protein domain, based on the cell-surface adhesin HMW1A, is preferentially recognized by antibodies from sera of an MS patient subpopulation. In conclusion the hyperglucosylated adhesin is the first example of an N-glucosylated native antigen that can be considered a relevant candidate for triggering pathogenic antibodies in MS.

Structurally defined materials on the nanometer length-scale have been historically the most challenging to rationally construct and the most difficult to structurally analyze. Sequence-specific biomolecules, i.e., proteins and nucleic acids, have advantages as design elements for construction of these types of nano-scale materials in that correlations can be drawn between sequence and higher order structure, potentially affording ordered assemblies in which functional properties can be controlled through the progression of structural hierarchy encoded at the molecular level. However, the predictable design of self-assembled structures requires precise structural control of the interfaces between peptide subunits (protomers). In contrast to the robustness of protein tertiary structure, quaternary structure has been postulated to be labile with respect to mutagenesis of residues located at the protein-protein interface. We have employed simple self-assembling peptide systems to interrogate the concept of designability of interfaces within the structural context of nanotubes and nanosheets. These peptide systems provide a framework for understanding how minor sequence changes in evolution can translate into very large changes in supramolecular structure, which provides significant evidence that the designability of protein interfaces is a critical consideration for control of supramolecular structure in self-assembling systems.

Hydrogels made of small peptides, especially di- and tripeptides, are particularly attractive owing to simple synthetic procedures, chemical variability, and potential for introduction biological functionality. The gelation of this type of peptides is usually driven by the cooperative effect of several weak intermolecular interactions such as hydrogen bonding, hydrophobic and aromatic-interactions. The main limitation of peptide hydrogels for biomedical applications is their susceptibility to enzymatic hydrolysis. A well established strategy to provide peptides and proteins with proteolytic stability is the replacement of natural amino acids by non-proteinogenic analogues such as D-amino acids, ß-amino acids or dehydroamino acids.

Small peptides with dehydroamino acids residues and N-capped with bulky aromatic moieties constitute an important class of hydrogelators that resists proteolysis. The dehydropeptides building blocks can be easily obtained from the corresponding peptides with β-hydroxyamino acid residues and give hydrogels at low critical gelation concentrations. Molecular dynamic simulations showed that the conformational constraints imposed by the dehydroamino acid residues are beneficial to the peptide self-assembly process into supramolecular structures. Preliminary studies using photophysical methods showed that these materials can be used as efficient nanocarriers of several antitumoral drugs.

Hydrogels obtained from small dehydropeptides can be considered as novel nano-pharmaceuticals since they ally proteolytic stability to potential intrinsic biological activity and drug delivery capability.

Supramolecular hydrogels, formed via intermolecular interactions in water, are emerging as a new type of versatile soft materials to be applied in many areas, such as biomedical applications, catalysis, food additives, and cosmetics. While most of the supramolecular hydrogels are homotypic (i.e., one type of building blocks), heterotypic supramolecular hydrogels are less explored, but may offer unique advantages. This talk discuss supramolecular hydrogels that consist of more than one type building blocks (i.e., heterotypic) to illustrate the promises and challenges of heterotypic supramolecular hydrogels as soft biomaterials. First, we discuss the driving force for producing heterotypic supramolecular hydrogels. Second, we introduce the general methods for triggering heterotypic supramolecular hydrogels. Third, we report an example of two complementary pentapeptides from a beta-sheet motif of a protein self-assemble to form beta-sheet like structures upon being mixed in water.  Although beta-sheet is a common secondary structure formed by certain segments of peptides in proteins, the isolated segments by themselves usually are unable to maintain the original secondary structures due to the lack of the conformation restriction provided by the proteins. By promoting the pentapeptides transform from alpha-helix to beta-sheet conformation, the self-assembly results in supramolecular hydrogels. This talk will illustrate a bioinspired way to generate supramolecular peptide nanofibers, a class of bioactive entities, with predefined secondary structures by a rational design that uses protein structures as the blueprint.

Membrane fusion in case of synaptic transmission is triggered by fusion proteins like SNARE (Soluble N-ethylmaleimide-Sensitive Factor Attachment Protein Receptor) proteins. A coiled-coil four-helix bundle is formed between SNARE proteins syntaxin-1A and SNAP-25 residing in the plasma membrane and the SNARE protein synaptobrevin residing in the membrane of synaptic vesicles, forcing the two merging membranes in close proximity. The precise mechanism of SNARE mediated membrane fusion, e.g. the role of transmembrane domains of synaptobrevin (Syb) and syntaxin-1A (Sx), is still under debate. Therefore, fusion experiments are described using vesicles reconstituted with artificial SNARE mimicking model systems, thereby, simplifying the SNARE assembly reaction and allowing systematic structural variations. SNARE analogous model systems based on transmembrane peptides and covalently linked recognition motifs like coiled-coil forming peptides or peptide nucleic acids (PNAs) are described. The PNA recognition motif is especially suited to control the directionality of the recognition process using caging groups thereby controlling vesicle docking and fusion. In addition, an interdependence between the recognition process and the peptide helix transmembrane domain was postulated with respect to vesicle docking and fusion efficiency. The transmembrane peptide and especially the charge of C-terminus have significant influence on the fusion mechanism.

The use of peptides as therapeutics is undergoing a renewed enthusiasm owing to a notable expansion of the number of marketing approvals in the recent years. Current successes in the development of peptides as therapeutics are likely to spur additional growth in this sector. However, clinical uses of peptide-based medicines are still challenged by short in vivo life time and low stability. Chemical modifications, such as substitutions, acylation and PEGylation, have compensated some but not all of their promises. From the development point of view, the reduced size of peptides contrasted with larger biologics upsurge to different formulation hurdles, mostly towards to chemical, conformational stability and aggregation. These unique features result in challenges of attaining satisfactory chemical and physical stability through the manufacturing process and during shelf-life. Hence, this complex feature also makes them some of the most thought-provoking molecules to design, formulate and deliver. Strategies to limit the aggregation of peptides during storage are likely to benefit from the recent surge of interest in peptide fibrillation. This talk focuses on a brief overview of formulation issues and the approaches to mitigate some development obstacles associated with peptide therapeutics self-assembly and fibrillation. The presenter also emphasizes opportunities for the thoughtful application of peptide self-assembly to develop long acting peptide drugs.

Molecular self-association plays a pivotal role in chemical, biological and material sciences. Peptides with suitable functionalities are good candidates for assembly by using various non-covalent interactions including hydrogen bonding, pi-pi stacking, electrostatic, solvophobic and others. Under a suitable situation, a peptide can be self-assembled to form a micro/nano-fibrillar network structure occupied by a large amount of solvent molecules (water/organic solvent) and this forms a soft material called gel. It is interesting to control the assembly of designer oligopeptides to make useful gels and also to explore fascinating applications of these gels.

These gels were applied to perform a variety of functions including carriers of drugs and other biologically active molecules. Recently, a peptide-based thxiotropic, proteolytically stable hydrogel has been designed and constructed and it has shown a remarkable antibacterial properties against Gram-negative bacteria. Moreover, it has shown biocompatibility with human red blood cells and human fibroblast cells. Another interesting study demonstrates peptide based soft biomaterials for anti-cancer drug release and modulation of stiffness, drug release capacity and proteolytic stability of these hydrogels by incorporating D-amino acid residue(s).

Aggregation of Amyloid-β peptide (Aβ) into small oligomers, and into the amyloid fibrils associated with senile plaques, is a key event in the pathogenesis of Alzheimer’s disease.  The group have investigated the effects of nanoliposomes decorated with the retro-inverso peptide RI-OR2-TAT (Ac-rGffvlkGrrrrqrrkkrGy-NH2) on the aggregation and toxicity of Aβ. This retro-inverted peptide has been shown to bind to Aβ monomers and to prevent their assembly into oligomers and β-pleated sheet fibrils. Remarkably low concentrations of Peptide Inhibitory NanoParticles (PINPs) were required to inhibit the formation of Aβ oligomers and fibrils in vitro, with 50% inhibition occurring at a molar ratio of ~1:2000 of liposome-bound RI-OR2-TAT to Aβ. PINPs also bound to Aβ with high affinity (Kd = 13.2 - 50 nM), rescued SHSY-5Y cells from the toxic effects of pre-aggregated Aβ, crossed an in vitro blood-brain-barrier model (hCMEC/D3 cell monolayer), entered the brains of C57/BL6 mice, and protected against memory loss in APPSWE transgenic mice in a novel object recognition test. As the most potent Aβ aggregation inhibitor that has been tested so far, the group propose to develop PINPs as a potential disease-modifying treatment for slowing progression of Alzheimer’s disease.

This talk outlines the efforts by the group to explore the use of peptides and proteins as building blocks for the synthesis of microcapsules. The group use the self-assembly of polypeptide chains into nanofibrils to define the structure of these materials on the nanoscale, and exploit microfluidics to determine their micron scale morphology. Such capsules can be used for the stabilisation, storage and release of sensitive materials, in particular aggregation prone antibodies. Moreover, we explore the use of peptide self-assembly within microdroplets as the basis of active materials and demonstrate chemo-mechanical actuation in such systems.