Identification of the minimum pharmacophore of lipid–phosphatidylserine (PS) binding peptide–peptoid hybrid PPS1D1

We previously reported a unique peptide–peptoid hybrid, PPS1 that specifically recognizes lipid–phosphatidylserine (PS) and a few other negatively charged phospholipids, but not neutral phospholipids, on the cell membrane. The dimeric version of PPS1, i.e., PPS1D1 triggers strong cancer cell cytotoxicity and has been validated in lung cancer models both in vitro and in vivo. Given that PS and other negatively charged phospholipids are abundant in almost all tumor microenvironments, PPS1D1 is an attractive drug lead that can be developed into a globally applicable anti-cancer agent. Therefore, it is extremely important to identify the minimum pharmacophore of PPS1D1. In this study, we have synthesized alanine/sarcosine derivatives as well as truncated derivatives of PPS1D1. We performed ELISA-like competitive binding assay to evaluate the PS-recognition potential and standard MTS cell viability assay on HCC4017 lung cancer cells to validate the cell cytotoxicity effects of these derivatives. Our studies indicate that positively charged residues at the second and third positions, as well as four hydrophobic residues at the fifth through eighth positions, are imperative for the binding and activity of PPS1D1. Methionine at the first position was not essential, whereas the positively charged Nlys at the fourth position was minimally needed, as two derivatives that were synthesized replacing this residue were almost as active as PPS1D1.     

Structural characterization of cationic lipid–tRNA complexes

Despite considerable interest and investigations on cationic lipid–DNA complexes, reports on lipid–RNA interaction are very limited. In contrast to lipid–DNA complexes where lipid binding induces partial B to A and B to C conformational changes, lipid–tRNA complexation preserves tRNA folded state. This study is the first attempt to investigate the binding of cationic lipid with transfer RNA and the effect of lipid complexation on tRNA aggregation and condensation. We examine the interaction of tRNA with cholesterol (Chol), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), dioctadecyldimethylammoniumbromide (DDAB) and dioleoylphosphatidylethanolamine (DOPE), at physiological condition, using constant tRNA concentration and various lipid contents. FTIR, UV-visible, CD spectroscopic methods and atomic force microscopy (AFM) were used to analyze lipid binding site, the binding constant and the effects of lipid interaction on tRNA stability, conformation and condensation. Structural analysis showed lipid–tRNA interactions with G–C and A–U base pairs as well as the backbone phosphate group with overall binding constants of K_Chol = 5.94 (± 0.8) × 10⁴ M^–1, K_DDAB = 8.33 (± 0.90) × 10⁵ M^–1, K_DOTAP = 1.05 (± 0.30) × 10⁵ M^–1 and K_DOPE = 2.75 (± 0.50) × 10⁴ M^–1. The order of stability of lipid–tRNA complexation is DDAB > DOTAP > Chol > DOPE. Hydrophobic interactions between lipid aliphatic tails and tRNA were observed. RNA remains in A-family structure, while biopolymer aggregation and condensation occurred at high lipid concentrations.     

Structural features of the tmRNA–ribosome interaction

Trans-translation is a process which switches the synthesis of a polypeptide chain encoded by a nonstop messenger RNA to the mRNA-like domain of a transfer-messenger RNA (tmRNA). It is used in bacterial cells for rescuing the ribosomes arrested during translation of damaged mRNA and directing this mRNA and the product polypeptide for degradation. The molecular basis of this process is not well understood. Earlier, we developed an approach that allowed isolation of tmRNA–ribosomal complexes arrested at a desired step of tmRNA passage through the ribosome. We have here exploited it to examine the tmRNA structure using chemical probing and cryo-electron microscopy tomography. Computer modeling has been used to develop a model for spatial organization of the tmRNA inside the ribosome at different stages of trans-translation.     

Control and role of pH in peptide–lipid interactions in oriented membrane samples

To understand the molecular mechanisms of amphiphilic membrane-active peptides, one needs to study their interactions with lipid bilayers under ambient conditions. However, it is difficult to control the pH of the sample in biophysical experiments that make use of mechanically aligned multilamellar membrane stacks on solid supports. HPLC-purified peptides tend to be acidic and can change the pH in the sample significantly. Here, we have systematically studied the influence of pH on the lipid interactions of the antimicrobial peptide PGLa embedded in oriented DMPC/DMPG bilayers. Using solid-state NMR (³¹P, ²H, ¹⁹F), both the lipid and peptide components were characterized independently, though in the same oriented samples under typical conditions of maximum hydration. The observed changes in lipid polymorphism were supported by DSC on multilamellar liposome suspensions. On this basis, we can present an optimized sample preparation protocol and discuss the challenges of performing solid-state NMR experiments under controlled pH. DMPC/DMPG bilayers show a significant up-field shift and broadening of the main lipid phase transition temperature when lowering the pH from 10.0 to 2.6. Both, strongly acidic and basic pH, cause a significant degree of lipid hydrolysis, which is exacerbated by the presence of PGLa. The characteristic re-alignment of PGLa from a surface-bound to a tilted state is not affected between pH of 7 to 4 in fluid bilayers. On the other hand, in gel-phase bilayers the peptide remains isotropically mobile under acidic conditions, displays various co-existing orientational states at pH 7, and adopts an unknown structural state at basic pH.     

Binding of Aβ peptide creates lipid density depression in DMPC bilayer

Using isobaric–isothermal replica exchange molecular dynamics and all-atom explicit water model we study the impact of Aβ monomer binding on the equilibrium properties of DMPC bilayer. We found that partial insertion of Aβ peptide into the bilayer reduces the density of lipids in the binding “footprint” and indents the bilayer thus creating a lipid density depression. Our simulations also reveal thinning of the bilayer and a decrease in the area per lipid in the proximity of Aβ. Although structural analysis of lipid hydrophobic core detects disordering in the orientations of lipid tails, it also shows surprisingly minor structural perturbations in the tail conformations. Finally, partial insertion of Aβ monomer does not enhance water permeation through the DMPC bilayer and even causes considerable dehydration of the lipid–water interface. Therefore, we conclude that Aβ monomer bound to the DMPC bilayer fails to perturb the bilayer structure in both leaflets. Limited scope of structural perturbations in the DMPC bilayer caused by Aβ monomer may constitute the molecular basis of its low cytotoxicity.     

NMR structure of the water soluble Aβ17–34 peptide

Alzheimer''s disease is the most common neurodegenerative disorder in the world. Its most significant symptoms are memory loss and decrease in cognition. Alzheimer''s disease is characterized by aggregation of two proteins in the brain namely Aβ (amyloid β) and tau. Recent evidence suggests that the interaction of soluble Aβ with nAChR (nicotinic acetylcholine receptors) contributes to disease progression. In this study, we determine the NMR structure of an Aβ_17–34 peptide solubilized by the addition of two glutamic acids at each terminus. Our results indicate that the Aβ peptide adopts an α-helical structure for residues 19–26 and 28–33. The α-helical structure is broken around residues S26, N27 and K28, which form a kink in the helical conformation. This α-helix was not described earlier in an aqueous solution without organic solvents, and at physiological conditions (pH 7). These data are in agreement with Aβ adopting an α-helical conformation in the membrane before polymerizing into amyloid β-sheets and provide insight into the intermediate state of Aβ in Alzheimer''s disease.     

Evaluation of the Influence of thiosemicarbazone–triazole hybrids on genes implicated in lipid oxidation and accumulation as potential anti-obesity agents

A series of thiosemicarbazone–triazole hybrids 1a–h are efficiently synthesised and evaluated for their influence on the expression of genes, cpt-1, acc-1 and pgc-1, which are essential in lipid metabolism. The test results show that hybrids 1c and 1g exhibited relatively high influence on the expression of cpt-1 and pgc-1 and suppression of acc-1 as desired.     

Structural features of split and unsplit βαβ-units

In this study, 1064 nonhomologous “unsplit”, “one-strand split” and “two-strand split” right-handed βαβ-units having standard α-helices and loops up to seven residues in length have been analyzed. It was found that the α-helices in these kinds of βαβ-units have different distributions of the hydrophobic and hydrophilic amino acid residues along the chain. In the unsplit βαβ-units, most α-helices have hydrophobic residues in positions N4-N7-N8-N11 or N6-N7-N10, where N1 is the first N-terminal residue. In the one-strand split βαβ-units, most α-helices have hydrophobic residues in positions N4-N7-N8-N11 and those in two-strand split βαβ-units in positions N4-N5-N8-N12. On the other hand, in all kinds of βαβ-units, there are commonly occurring hydrophobic stripes of type C4-C7-C8 at the C-terminal parts of the α-helices. As a rule, the C- and N-terminal hydrophobic stripes overlap and the extent of their overlapping determine the length of α-helices.     

Insertion into lipid bilayer of truncated pHLIP® peptide

The investigation of pH-dependent membrane-associated folding has both fundamental interest and practical applications for targeting of acidic tumors and specific delivery of therapeutic molecules across membrane of cancer cells. We and others investigated molecular mechanism and medical uses of class of water soluble membrane peptides, pH (Low) Insertion Peptides (pHLIP^® peptides). Here we employed optical spectroscopy methods to study interactions of the truncated pHLIP^® peptide (Short pHLIP^®) with lipid bilayer of membrane. Tryptophan fluorescence, CD and OCD data indicate on pH-triggered formation of transmembrane helical structure. Dual quenching and FRET assays demonstrated that Short pHLIP^® peptide spans lipid bilayer of membrane similar to Long pHLIP^® peptides. Truncated pHLIP^® peptides with multiple charged and protonatable residues in their sequences potentially can make these peptides to be less hydrophobic compared to Long pHLIP^® peptides, and might have utility in tumor imaging, and potentially, in pH-regulated cytoplasmic delivery of moderately hydrophobic drugs.     

Structural study of the β-hairpin marine antimicrobial peptide arenicin-2 in PC/PG lipid bilayers by fourier transform infrared spectroscopy

Arenicin-2 is a 21-residue β-hairpin antimicrobial peptide isolated from the marine lugworm Arenicola marina. The structure of this cationic peptide in partly charged lipid membrane made of PC/PG (7: 3) was studied by FTIR, CD, and Trp fluorescence spectroscopies. FTIR spectra of arenicin in amide I region were analyzed using curve-fitting and second derivative procedures. The FTIR data for the peptide in PC/PG liposomes were compared with the data obtained in anionic SDS micelles where arenicin forms a dimer stabilized by parallel association of two β-hairpins according to previous NMR spectroscopy studies [Ovchinnikova et al., Biopolymers, 2007, vol. 89, pp. 455–464; Shenkarev et al., Biochemistry, 2011, vol. 50, pp. 6255–6265]. The results obtained in present work indicate that arenicin forms the dimeric structure in partly charged PC/PG lipid membrane. This finding is discussed in relation to interpretation of low-conducting pores observed for arenicin in negatively charged membranes.     

Cholesterol inhibits the insertion of the Alzheimer’s peptide Aβ(25–35) in lipid bilayers

The physiological relationship between brain cholesterol content and the action of amyloid β (Aβ) peptide in Alzheimer’s disease (AD) is a highly controversially discussed topic. Evidences for modulations of the Aβ/membrane interaction induced by plasma membrane cholesterol have already been observed. We have recently reported that Aβ(25–35) is capable of inserting in lipid membranes and perturbing their structure. Applying neutron diffraction and selective deuteration, we now demonstrate that cholesterol alters, at the molecular level, the capability of Aβ(25–35) to penetrate into the lipid bilayers; in particular, a molar weight content of 20% of cholesterol hinders the intercalation of monomeric Aβ(25–35) completely. At very low cholesterol content (about 1% molar weight) the location of the C-terminal part of Aβ(25–35) has been unequivocally established in the hydrocarbon region of the membrane, in agreement with our previous results on pure phospholipids membrane. These results link a structural property to a physiological and functional behavior and point to a therapeutical approach to prevent the AD by modulation of membrane properties.     

The role of membrane thickness in charged protein–lipid interactions

Charged amino acids are known to be important in controlling the actions of integral and peripheral membrane proteins and cell disrupting peptides. Atomistic molecular dynamics studies have shed much light on the mechanisms of membrane binding and translocation of charged protein groups, yet the impact of the full diversity of membrane physico-chemical properties and topologies has yet to be explored. Here we have performed a systematic study of an arginine (Arg) side chain analog moving across saturated phosphatidylcholine (PC) bilayers of variable hydrocarbon tail length from 10 to 18 carbons. For all bilayers we observe similar ion-induced defects, where Arg draws water molecules and lipid head groups into the bilayers to avoid large dehydration energy costs. The free energy profiles all exhibit sharp climbs with increasing penetration into the hydrocarbon core, with predictable shifts between bilayers of different thickness, leading to barrier reduction from 26 kcal/mol for 18 carbons to 6 kcal/mol for 10 carbons. For lipids of 10 and 12 carbons we observe narrow transmembrane pores and corresponding plateaus in the free energy profiles. Allowing for movements of the protein and side chain snorkeling, we argue that the energetic cost for burying Arg inside a thin bilayer will be small, consistent with recent experiments, also leading to a dramatic reduction in pK_a shifts for Arg. We provide evidence that Arg translocation occurs via an ion-induced defect mechanism, except in thick bilayers (of at least 18 carbons) where solubility-diffusion becomes energetically favored. Our findings shed light on the mechanisms of ion movement through membranes of varying composition, with implications for a range of charged protein–lipid interactions and the actions of cell-perturbing peptides. This article is part of a Special Issue entitled: Membrane protein structure and function.     

Importance of phospholipid bilayer integrity in the analysis of protein–lipid interactions

The integrity of supported phospholipid bilayer membranes is of crucial importance for the investigation of lipid–protein interactions. Therefore we recorded the formation of supported membranes on SiO₂ and mica by quartz crystal microbalance and controlled the integrity by atomic force microscopy. This study aims to analyze how membrane defects affect protein–lipid interactions. The experiments focused on a lipid mixture of POPC/DOPC/Chol/POPS/PI(4,5)P₂ (37:20:20:20:3) and the binding of the peripheral membrane associated protein annexin A2. We found that formation of a continuous undisturbed bilayer is an indispensable precondition for a reliable determination and quantification of lipid–protein-interactions. If membrane defects were present, protein adsorption causes membrane disruption and lipid detachment on a support thus leading to false determination of binding constants. Our results obtained for PI(4,5)P₂ and cholesterol containing supported membranes yield new knowledge to construct functional surfaces that may cover nanoporous substrates, form free standing membranes or may be used for lab-on-a-chip applications.     

Oligomeric amyloid-β peptide affects the expression of genes involved in steroid and lipid metabolism in primary neurons

Amyloid-β peptide (Aβ) is the principal component of plaques in the brains of patients with Alzheimer's disease (AD), and the most toxic form of Aβ may be as soluble oligomers. We report here the results of a microarray study of gene expression profiles in primary mouse cortical neurons in response to oligomeric Aβ(1-42). A major and unexpected finding was the down-regulation of genes involved in the biosynthesis of cholesterol and other steroids and lipids (such as Fdft1, Fdps, Idi1, Ldr, Mvd, Mvk, Nsdhl, Sc4mol), the expression of which was verified by quantitative real-time RT-PCR (qPCR). The ATP-binding cassette gene Abca1, which has a major role in cholesterol transport in brain and other tissues and has been genetically linked to AD, was notably up-regulated. The possible involvement of cholesterol and other lipids in Aβ synthesis and action in Alzheimer's disease has been studied and debated extensively but remains unresolved. These new data suggest that Aβ may influence steroid and lipid metabolism in neurons via multiple gene-expression changes.     

Ratiometric imaging of gastrodermal lipid bodies in coral–dinoflagellate endosymbiosis

Cnidaria–dinoflagellate endosymbiosis is the phenomenon of autotrophic symbionts living inside the gastrodermal cells of their animal hosts. The molecular mechanism that regulates this association remains unclear. Using quantitative microscopy, we now provide evidence that the dynamic lipid changes in gastrodermal “lipid bodies” (LBs) reflect the symbiotic status of the host cell and its symbiont in the hermatypic coral Euphyllia glabrescens. By dual-emission ratiometric imaging with a solvatochromic fluorescent probe, Nile red (9-diethylamino-5H-benzo[α]phenoxazine-5-one), we showed that the in situ distribution of polar versus neutral lipids in LBs in living gastrodermal cells and symbionts can be analyzed. The ratio of Nile red fluorescence at red (R) versus green (G) wavelength region (i.e., R/G ratio) correlated with the relative molar ratio of polar (P) versus neutral (NP) lipids (i.e., P/NP ratio). The R/G ratio in host LBs increased after bleaching, indicating a decrease in neutral lipid accumulation in gastrodermal cells. On the other hand, neutral lipid accumulation inside the symbiont LBs resulted in gradual decreases of the R/G ratio as a result of bleaching. In comparison with the bleaching event, there was no relative lipid concentration change in host LBs under continual light or dark treatments as shown by insignificant R/G ratio shift. Patterns of R/G ratio shift in symbiont LBs were also different between corals undergoing bleaching and continual light/dark treatment. In the latter, there was little lipid accumulation in symbionts, with no resulting R/G ratio decrease. These results, demonstrating that the symbiotic status positively correlated with morphological and compositional changes of lipid bodies, not only highlight the pivotal role of LBs, but also implicate an involvement of lipid trafficking in regulating the endosymbiosis.