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[Display omitted] • Lipidating salmon calcitonin (sCal) alters its biophysical characteristics, including oligomer size, hydrophobicity and membrane activity. • Fluorescent imaging of single GUVs determines peptide mediated reporter dye influx behavior as either graded or All-or-None mechanism. • Modulating the number of hydrocarbons constituting the lipidation moieties determines the membrane permeation mechanism. • By increasing the lipid chain length lipidated of sCal goes from displaying smaller membrane perturbations to a peptide pore formation mechanism. • Effective membrane translocation of lipidated salmon calcitonin requires a peptide mediated pore forming mechanism. A widespread strategy to increase the transport of therapeutic peptides across cellular membranes has been to attach lipid moieties to the peptide backbone (lipidation) to enhance their intrinsic membrane interaction. Efforts in vitro and in vivo investigating the correlation between lipidation characteristics and peptide membrane translocation efficiency have traditionally relied on end-point read-out assays and trial-and-error-based optimization strategies. Consequently, the molecular details of how therapeutic peptide lipidation affects it's membrane permeation and translocation mechanisms remain unresolved. Here we employed salmon calcitonin as a model therapeutic peptide and synthesized nine double lipidated analogs with varying lipid chain lengths. We used single giant unilamellar vesicle (GUV) calcein influx time-lapse fluorescence microscopy to determine how tuning the lipidation length can lead to an All-or-None GUV filling mechanism, indicative of a peptide mediated pore formation. Finally, we used a GUVs-containing-inner-GUVs assay to demonstrate that only peptide analogs capable of inducing pore formation show efficient membrane translocation. Our data provided the first mechanistic details on how therapeutic peptide lipidation affects their membrane perturbation mechanism and demonstrated that fine-tuning lipidation parameters could induce an intrinsic pore-forming capability. These insights and the microscopy based workflow introduced for investigating structure–function relations could be pivotal for optimizing future peptide design strategies. [ABSTRACT FROM AUTHOR]

Lipoprotein signal peptidase (Lsp) is a transmembrane aspartic acid protease with a pivotal role in the bacterial lipoprotein maturation pathway. Despite the universal use of Lsp across the Bacterial Kingdom and its potential as an antibiotic target, the substrate recognition patterns of Lsp are poorly understood. Here, we investigated the substrate recognition and biochemical properties of Lsp from Gram− (Escherichia coli) and Gram+ (Streptococcus pyogenes) bacteria, using synthetic peptide‐based FRET reporters. Didecanoyl glycerol was found to be an optimal lipid length, and Lsp demonstrated exclusive enantio‐selectivity for the (R)‐form of the diacylglycerol. Our study will facilitate the iterative optimization of in vitro Lsp assays, as well as provide the first chemical interrogation into the substrate scope of Lsp. [ABSTRACT FROM AUTHOR]

An overall investigation was made of the cell morphology, adhesion, viability, and proliferation of human fibroblast cells on surfaces functionalized with peptide and peptide amphiphiles. We compared surfaces in which the RGD peptide was immobilized covalently onto silicon with those in which lipidated versions of the RGD peptide were hydrophobically attached to the alkylated silicon surfaces. The hydrophobically attached peptide amphiphile on alkylated silicon surfaces produce structures that are somewhat akin to the structure of a cell membrane. Scanning electron microscopy (SEM) and Laser scanning confocal microscopy (LSCM) were used to characterize the seeding human fibroblast cells on all prepared surfaces. Surfaces were also evaluated with a methyl tetrazole sulfate (MTS) assay to compare the proliferation ability. Cell-substrate interactions were examined through cell adhesion assay. Peptide-amphiphile modified surfaces exhibited substantially superior cellular responses compared to those on the covalently immobilized peptide. It was also shown that the length of alkyl tail in lipidated peptides may influence cellular response. Hydrophobically attached peptide amphiphiles on alkylated silicon surfaces may suggest new biomimetic platforms for further studies of the interaction between cells and extracellular matrix. [ABSTRACT FROM AUTHOR]

Hybrid incretin peptides are a new generation of drugs for the treatment of diabetes and obesity. Despite their biological potency, the effectiveness of these peptides as drugs is limited by their short circulation time in blood (typically within minutes). In this work, we show that lipid conjugated forms of a GLP-1/GIP/glucagon hybrid peptides stay in circulation for hours. We studied the oligomerization and albumin-binding of the unconjugated hybrid peptide as well as its lipidated variants. These lipidated peptides differ in the N-terminal mutation, the position of lipidation and the linkage to lipid. We found that these lipidated peptides form stable oligomers at concentrations above 1 mg/mL. This concentration range is relevant to formulation and storage of the peptides. We observed no binding between the peptide oligomers and human serum albumin. However, at the expected therapeutic concentration range (~ 10–100 ng/mL), the oligomers dissociate into monomers. The monomers of lipidated peptides bind to albumin. We have determined the dissociation constants of binding between the lipidated peptides and serum albumin. The dissociation constants of albumin-binding of our lipidated peptides are all very close and similar to that of the fatty acid binding of albumin. Our findings suggest that the monomeric lipidated peptides bind to HSA mainly by the fatty acid chain. Therefore, albumin binding is likely to be a universal mechanism of the prolonged circulating duration of lipidated pharmaceutical peptides. [ABSTRACT FROM AUTHOR]

High systemic toxicity of antimicrobial peptides (AMPs) limits their clinical application to the treatment of topical infections; in parenteral systemic application of AMPs the problem of hemolysis is one of the first to be tackled. We now show that the selectivity of lipidated short synthetic AMPs can be optimized substantially by reducing their hemolytic activity without affecting their activity against methicillin resistant Staphylococcus aureus (MRSA). In order to identify the optimized peptides, two sets of 32 diastereomeric H-dArg-WRWRW-lLys(C(O)CnH2n+1)-NH2 (n = 7 or 9) peptides were prepared using a split-split procedure to perform a systematic l-to-d exchange scan on the central WRWRW-fragment. Compared to the all-l C8-lipidated lead sequence, diastereomeric peptides had very similar antibacterial properties, but were over 30 times less hemolytic. We show that the observed hemolysis and antibacterial activity is affected by both differences in lipophilicity of the different peptides and specific combinations of l- and d-amino acid residues. This study identified several peptides that can be used as tools to precisely unravel the origin of hemolysis and thus help to design even further optimized nontoxic very active short antibacterial peptides. [ABSTRACT FROM AUTHOR]

Abstract: Lipidation of a short hydrophilic peptide has the aim to make the molecule amphiphilic, which improves its insertion into lipid monolayer and at the same time, the tendency to self-assembly. These both properties of two positively charged, hepatitis G (GBV-C/HGV) related lipidated peptides—palmitic acid derivatives of the fragments: 505–514 and the 513–522 of the NS3 protein (respectively Palmitoyl-SAELSMQRRG and Palmitoyl-RGRTGRGRSG) were studied. First, using transmission electron microscope (TEM) and atomic force microscope (AFM) the tendency to self-assembly in water solution was examined. Both techniques confirmed the formation of fibrous aggregates of Palmitoyl-SAELSMQRRG in water solution. At the same conditions, any fibrous aggregates of Palmitoyl-RGRTGRGRSG were detected neither by TEM nor by AFM. Insertion of the lipidated peptides into phospholipids monolayer formed by zwitterionic 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or negatively charged 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DPPG) was investigated. Monolayers prepared by Langmuir–Blodgett method were visualized by AFM. The presence of lipidated peptides in phospholipid monolayers produced changes in the monolayers and different morphologies of the monolayers were obtained for each of the lipidated peptides. [Copyright &y& Elsevier]

Abstract: In vitro immunization with HCV lipopeptide aa 20–44 results in antigen-specific T cells. Here, we studied whether presentation of the lipopeptide by dendritic cells (DC) results in enhanced T cell functions. DC-immunized T cells showed significantly augmented proliferation and IFN-γ secretion upon HCV-specific re-stimulation. This effect was related to significantly increased expression of TLR2 on DC-immunized CD4+ T cells and required TLR2 triggering during re-stimulation. In contrast, numbers of HLA-A2-pentamer-positive CD8+ T cells and granzyme B-secreting T cells remained unchanged. Thus, up-regulation of TLR2 during immunization with DC enhances functions of CD4+ T cells but not CD8+ T cells. [Copyright &y& Elsevier]