Impacts of Hydrophobic Mismatch on Antimicrobial Peptide Efficacy and Bilayer Permeabilization.

Antimicrobial resistance continues to be a major threat to world health, with the continued emergence of resistant bacterial strains. Antimicrobial peptides have emerged as an attractive option for the development of novel antimicrobial compounds in part due to their ubiquity in nature and the general lack of resistance development to this class of molecules. In this work, we analyzed the antimicrobial peptide C18G and several truncated forms for efficacy and the underlying mechanistic effects of the sequence truncation. The peptides were screened for antimicrobial efficacy against several standard laboratory strains, and further analyzed using fluorescence spectroscopy to evaluate binding to model lipid membranes and bilayer disruption. The results show a clear correlation between the length of the peptide and the antimicrobial efficacy. Furthermore, there is a correlation between peptide length and the hydrophobic thickness of the bilayer, indicating that hydrophobic mismatch is likely a contributing factor to the loss of efficacy in shorter peptides.

Exploratory analysis reveals arthropod consumption in 10 lemur species using DNA metabarcoding.

Arthropods (insects, spiders, etc.) can fulfill major nutritional requirements for primates, particularly in terms of proteins, fats, vitamins, and minerals. Yet, for many primate species we know very little about the frequency and importance of arthropod consumption. Traditional methods for arthropod prey identification, such as behavioral observations and fecal dissections, offer limited taxonomic resolution and, as a result, underestimate true diversity. Metabarcoding arthropod DNA from primate fecal samples provides a promising but underused alternative. Here, we inventoried arthropod prey diversity in wild lemurs by sequencing two regions of the CO1 gene. Samples were collected opportunistically from 10 species of lemurs inhabiting three national parks in southern Madagascar using a combination of focal animal follows and live trapping. In total, we detected arthropod DNA in 98 of the 170 fecal samples analyzed. Although all lemur species included in these analyses showed evidence of arthropod consumption, those within the family Cheirogaleidae appeared to consume the highest frequency and diversity of arthropods. To our knowledge, this study presents the first evidence of arthropod consumption in Phaner pallescens, Avahi peyrierasi, and Propithecus verreauxi, and identifies 32 families of arthropods as probable food items that have not been published as lemur dietary items to date. Our study emphasizes the importance of arthropods as a nutritional source and the role DNA metabarcoding can play in elucidating an animal's diet.

Analysis of Baboon IAPP Provides Insight into Amyloidogenicity and Cytotoxicity of Human IAPP.

The polypeptide hormone islet amyloid polypeptide (IAPP) forms islet amyloid in type 2 diabetes, a process which contributes to pancreatic ß-cell dysfunction and death. Not all species form islet amyloid, and the ability to do so correlates with the primary sequence. Humans form islet amyloid, but baboon IAPP has not been studied. The baboon peptide differs from human IAPP at three positions containing K1I, H18R, and A25T substitutions. The K1I substitution is a rare example of a replacement in the N-terminal region of amylin. The effect of this mutation on amyloid formation has not been studied, but it reduces the net charge, and amyloid prediction programs suggest that it should increase amyloidogenicity. The A25T replacement involves a nonconservative substitution in a region of IAPP that is believed to be important for aggregation, but the effects of this replacement have not been examined. The H18R point mutant has been previously shown to reduce aggregation in vitro. Baboon amylin forms amyloid on the same timescale as human amylin in vitro and exhibits similar toxicity toward cultured ß-cells. The K1I replacement in human amylin slightly reduces toxicity, whereas the A25T substitution accelerates amyloid formation and enhances toxicity. Photochemical cross-linking reveals that the baboon amylin, like human amylin, forms low-order oligomers in the lag phase of amyloid formation. Ion-mobility mass spectrometry reveals broadly similar gas phase collisional cross sections for human and baboon amylin monomers and dimers, with some differences in the arrival time distributions. Preamyloid oligomers formed by baboon amylin, but not baboon amylin fibers, are toxic to cultured ß-cells. The toxicity of baboon oligomers and lack of significantly detectable toxicity with exogenously added amyloid fibers is consistent with the hypothesis that preamyloid oligomers are the most toxic species produced during IAPP amyloid formation.

Analysis of Proline Substitutions Reveals the Plasticity and Sequence Sensitivity of Human IAPP Amyloidogenicity and Toxicity.

Pancreatic amyloid formation by the polypeptide IAPP contributes to ß-cell dysfunction in type 2 diabetes. There is a 1:1 correspondence between the ability of IAPP from different species to form amyloid in vitro and the susceptibility of the organism to develop diabetes. Rat IAPP is non-amyloidogenic and differs from human IAPP at six positions, including three proline replacements: A25P, S28P, and S29P. Incorporation of these proline residues into human IAPP leads to a non-amyloidogenic analogue that is used clinically. The role of the individual proline residues is not understood. We examine the three single and three double proline substitutions in the context of human IAPP. An S28P substitution significantly decreases amyloidogenicity and toxicity, while an S29P substitution has very modest effects despite being an identical replacement just one residue away. The consequences of the A25P substitution are between those of the two Ser to Pro substitutions. Double analogues containing an S28P replacement are less amyloidogenic and less toxic than the IAPPA25P S29P double analogue. Ion mobility mass spectrometry reveals that there is no correlation between the monomer or dimer conformation as reported by collision cross section measurements and the time to form amyloid. The work reveals both the plasticity of IAPP amyloid formation and the exquisite sequence sensitivity of IAPP amyloidogenicity and toxicity. The study highlights the key role of the S28P substitution and provides information that will aid in the rational design of soluble variants of IAPP. The variants studied here offer a system for further exploring features that control IAPP toxicity.

Analysis of the Role of the Conserved Disulfide in Amyloid Formation by Human Islet Amyloid Polypeptide in Homogeneous and Heterogeneous Environments.

Human islet amyloid polypeptide (hIAPP) is a hormone secreted from ß-cells in the Islets of Langerhans in response to the same stimuli that lead to insulin secretion. hIAPP plays an adaptive role in glucose homeostasis but misfolds to form insoluble, fibrillar aggregates in type II diabetes that are associated with the disease. Along the misfolding pathway, hIAPP forms species that are toxic to ß-cells, resulting in reduced ß-cell mass. hIAPP contains a strictly conserved disulfide bond between residues 2 and 7, which forms a small loop at the N-terminus of the molecule. The loop is located outside of the cross ß-core in all models of the hIAPP amyloid fibrils. Mutations in this region are rare, and the disulfide loop plays a role in receptor binding; however, the contribution of this region to the aggregation of hIAPP is not well understood. We define the role of the disulfide by analyzing a collection of analogues that remove the disulfide, by mutation of Cys to Ser, by reduction and modification of the Cys residues, or by deletion of the first seven residues. The cytotoxic properties of hIAPP are retained in the Cys to Ser disulfide-free mutant. Removal of the disulfide bond accelerates amyloid formation in all constructs, both in solution and in the presence of model membranes. Removal of the disulfide weakens the ability of hIAPP to induce leakage of vesicles consisting of POPS and POPC. Smaller effects are observed with vesicles that contain 40 mol % cholesterol, although N-terminal truncation still reduces the extent of leakage.

Role of Cationic Side Chains in the Antimicrobial Activity of C18G.

Antimicrobial peptides (AMPs) have been an area of great interest, due to the high selectivity of these molecules toward bacterial targets over host cells and the limited development of bacterial resistance to these molecules throughout evolution. The peptide C18G has been shown to be a selective, broad spectrum AMP with a net +8 cationic charge from seven lysine residues in the sequence. In this work, the cationic Lys residues were replaced with other natural or non-proteinogenic cationic amino acids: arginine, histidine, ornithine, or diaminopropionic acid. These changes vary in the structure of the amino acid side chain, the identity of the cationic moiety, and the pKa of the cationic group. Using a combination of spectroscopic and microbiological methods, the influence of these cationic groups on membrane binding, secondary structure, and antibacterial activity was investigated. The replacement of Lys with most other cationic residues had, at most, 2-fold effects on minimal inhibitory concentration against a variety of Gram-positive and Gram-negative bacteria. However, the peptide containing His as the cationic group showed dramatically reduced activity. All peptide variants retained the ability to bind lipid vesicles and showed clear preference for binding vesicles that contained anionic lipids. Similarly, all peptides adopted a helical conformation when bound to lipids or membrane mimetics, although the peptide containing diaminopropionic acid exhibited a decreased helicity. The peptides exhibited a wider variety of activity in the permeabilization of bacterial membranes, with peptides containing Lys, Arg, or Orn being the most broadly active. In all, the antibacterial activity of the C18G peptide is generally tolerant to changes in the structure and identity of the cationic amino acids, yielding new possibilities for design and development of AMPs that may be less susceptible to immune and bacterial recognition or in vivo degradation.

Molecular Signature for Receptor Engagement in the Metabolic Peptide Hormone Amylin.

The pancreatic peptide hormone, amylin, plays a critical role in the control of appetite, and synergizes with other key metabolic hormones such as glucagon-like peptide 1 (GLP-1). There is opportunity to develop potent and long-acting analogues of amylin or hybrids between these and GLP-1 mimetics for treating obesity. To achieve this, interrogation of how the 37 amino acid amylin peptide engages with its complex receptor system is required. We synthesized an extensive library of peptides to profile the human amylin sequence, determining the role of its disulfide loop, amidated C-terminus and receptor "capture" and "activation" regions in receptor signaling. We profiled four signaling pathways with different ligands at multiple receptor subtypes, in addition to exploring selectivity determinants between related receptors. Distinct roles for peptide subregions in receptor binding and activation were identified, resulting in peptides with greater activity than the native sequence. Enhanced peptide activity was preserved in the brainstem, the major biological target for amylin. Interpretation of our data using full-length active receptor models supported by molecular dynamics, metadynamics, and supervised molecular dynamics simulations guided the synthesis of a potent dual agonist of GLP-1 and amylin receptors. The data offer new insights into the function of peptide amidation, how allostery drives peptide-receptor interactions, and provide a valuable resource for the development of novel amylin agonists for treating diabetes and obesity.

Evolutionary Adaptation and Amyloid Formation: Does the Reduced Amyloidogenicity and Cytotoxicity of Ursine Amylin Contribute to the Metabolic Adaption of Bears and Polar Bears?

Much of our knowledge of diabetes is derived from studies of rodent models. An alternative approach explores evolutionary solutions to physiological stress by studying organisms that face challenging metabolic environments. Polar bears eat an enormously lipid-rich diet without deleterious metabolic consequences. In contrast, transgenic rodents expressing the human neuropancreatic polypeptide hormone amylin develop hyperglycemia and extensive pancreatic islet amyloid when fed a high fat diet. The process of islet amyloid formation by human amylin contributes to ß-cell dysfunction and loss of ß-cell mass in type-2 diabetes. We show that ursine amylin is considerably less amyloidogenic and less toxic to ß-cells than human amylin, consistent with the hypothesis that part of the adaptation of bears to metabolic challenges might include protection from islet amyloidosis-induced ß-cell toxicity. Ursine and human amylin differ at four locations: H18R, S20G, F23L, and S29P. These are interesting from a biophysical perspective since the S20G mutation accelerates amyloid formation but the H18R slows it. An H18RS20G double mutant of human amylin behaves similarly to the H18R mutant, indicating that the substitution at position 18 dominates the S20G replacement. These data suggest one possible mechanism underpinning the protection of bears against metabolic challenges and provide insight into the design of soluble analogs of human amylin.

Time-resolved studies define the nature of toxic IAPP intermediates, providing insight for anti-amyloidosis therapeutics.

Islet amyloidosis by IAPP contributes to pancreatic ß-cell death in diabetes, but the nature of toxic IAPP species remains elusive. Using concurrent time-resolved biophysical and biological measurements, we define the toxic species produced during IAPP amyloid formation and link their properties to induction of rat INS-1 ß-cell and murine islet toxicity. These globally flexible, low order oligomers upregulate pro-inflammatory markers and induce reactive oxygen species. They do not bind 1-anilnonaphthalene-8-sulphonic acid and lack extensive ß-sheet structure. Aromatic interactions modulate, but are not required for toxicity. Not all IAPP oligomers are toxic; toxicity depends on their partially structured conformational states. Some anti-amyloid agents paradoxically prolong cytotoxicity by prolonging the lifetime of the toxic species. The data highlight the distinguishing properties of toxic IAPP oligomers and the common features that they share with toxic species reported for other amyloidogenic polypeptides, providing information for rational drug design to treat IAPP induced ß-cell death.

Islet Amyloid Polypeptide: Structure, Function, and Pathophysiology.

The hormone islet amyloid polypeptide (IAPP, or amylin) plays a role in glucose homeostasis but aggregates to form islet amyloid in type-2 diabetes. Islet amyloid formation contributes to ß-cell dysfunction and death in the disease and to the failure of islet transplants. Recent work suggests a role for IAPP aggregation in cardiovascular complications of type-2 diabetes and hints at a possible role in type-1 diabetes. The mechanisms of IAPP amyloid formation in vivo or in vitro are not understood and the mechanisms of IAPP induced ß-cell death are not fully defined. Activation of the inflammasome, defects in autophagy, ER stress, generation of reactive oxygen species, membrane disruption, and receptor mediated mechanisms have all been proposed to play a role. Open questions in the field include the relative importance of the various mechanisms of ß-cell death, the relevance of reductionist biophysical studies to the situation in vivo, the molecular mechanism of amyloid formation in vitro and in vivo, the factors which trigger amyloid formation in type-2 diabetes, the potential role of IAPP in type-1 diabetes, the development of clinically relevant inhibitors of islet amyloidosis toxicity, and the design of soluble, bioactive variants of IAPP for use as adjuncts to insulin therapy.

Analysis of the ability of pramlintide to inhibit amyloid formation by human islet amyloid polypeptide reveals a balance between optimal recognition and reduced amyloidogenicity.

The hormone human islet amyloid polypeptide (hIAPP or amylin) plays a role in glucose metabolism, but forms amyloid in the pancreas in type 2 diabetes (T2D) and is associated with ß-cell death and dysfunction in the disease. Inhibitors of islet amyloid have therapeutic potential; however, there are no clinically approved inhibitors, and the mode of action of existing inhibitors is not well understood. Rat IAPP (rIAPP) differs from hIAPP at six positions, does not form amyloid, and is an inhibitor of amyloid formation by hIAPP. Five of the six differences are located within the segment of residues 20-29, and three of them are Pro residues, which are well-known disruptors of ß-sheet structure. rIAPP is thus a natural example of a "ß-breaker inhibitor", a molecule that combines a recognition element with an entity that inhibits ß-sheet formation. Pramlintide (PM) is a peptide drug approved for use as an adjunct to insulin therapy for treatment of diabetes. PM was developed by introducing the three Pro substitutions found in rIAPP into hIAPP. Thus, it more closely resembles the human peptide than does rIAPP. Here we examine and compare the ability of rIAPP, PM, and a set of designed analogues of hIAPP to inhibit amyloid formation by hIAPP, to elucidate the factors that lead to effective peptide-based inhibitors. Our results reveal, for this class of molecules, a balance between the reduced amyloidogenicity of the inhibitory sequence on one hand and its ability to recognize hIAPP on the other.

Functional characterization of a melittin analog containing a non-natural tryptophan analog.

Tryptophan (Trp) is a naturally occurring amino acid, which exhibits fluorescence emission properties that are dependent on the polarity of the local environment around the Trp side chain. However, this sensitivity also complicates interpretation of fluorescence emission data. A non-natural analogue of tryptophan, ß-(1-azulenyl)-L-alanine, exhibits fluorescence insensitive to local solvent polarity and does not impact the structure or characteristics of several peptides examined. In this study, we investigated the effect of replacing Trp with ß-(1-azulenyl)-L-alanine in the well-known bee-venom peptide melittin. This peptide provides a model framework for investigating the impact of replacing Trp with ß-(1-azulenyl)-L-alanine in a functional peptide system that undergoes significant shifts in Trp fluorescence emission upon binding to lipid bilayers. Microbiological methods including assessment of the antimicrobial activity by minimal inhibitory concentration (MIC) assays and bacterial membrane permeability assays indicated little difference between the Trp and the ß-(1-azulenyl)-L-alanine-substituted versions of melittin. Circular dichroism spectroscopy showed both that peptides adopted the expected α-helical structures when bound to phospholipid bilayers and electrophysiological analysis indicated that both created membrane disruptions leading to significant conductance increases across model membranes. Both peptides exhibited a marked protection of the respective fluorophores when bound to bilayers indicating a similar membrane-bound topology. As expected, while fluorescence quenching and CD indicate the peptides are stably bound to lipid vesicles, the peptide containing ß-(1-azulenyl)-L-alanine exhibited no fluorescence emission shift upon binding while the natural Trp exhibited >10 nm shift in emission spectrum barycenter. Taken together, the ß-(1-azulenyl)-L-alanine can serve as a solvent insensitive alternative to Trp that does not have significant impacts on structure or function of membrane interacting peptides.

Poly(ethylene imine)s as antimicrobial agents with selective activity.

We report the structure-activity relationship in the antimicrobial activity of linear and branched poly(ethylene imine)s (L- and B-PEIs) with a range of molecular weights (MWs) (500-12,000). Both L- and B-PEIs displayed enhanced activity against Staphylococcus aureus over Escherichia coli. Both B- and L-PEIs did not cause any significant permeabilization of E. coli cytoplasmic membrane. L-PEIs induced depolarization of S. aureus membrane although B-PEIs did not. The low MW B-PEIs caused little or no hemolysis while L-PEIs are hemolytic. The low MW B-PEIs are less cytotoxic to human HEp-2 cells than other PEIs. However, they induced significant cell viability reduction after 24 h incubation. The results presented here highlight the interplay between polymer size and structure on activity.