Cell-penetrating peptides in protein mimicry and cancer therapeutics.

Extensive research has been undertaken in the pursuit of anticancer therapeutics. Many anticancer drugs require specificity of delivery to cancer cells, whilst sparing healthy tissue. Cell-penetrating peptides (CPPs), now well established as facilitators of intracellular delivery, have in recent years advanced to incorporate target specificity and thus possess great potential for the targeted delivery of anticancer cargoes.

Cell-Penetrating Peptides.

In this introductory chapter, we first define cell-penetrating peptides (CPPs), give short overview of CPP history and discuss several aspects of CPP classification. Next section is devoted to the mechanism of CPP penetration into the cells, where direct and endocytic internalization of CPP is explained. Kinetics of internalization is discussed more extensively, since this topic is not discussed in other chapters of this book.

Bioportide: an emergent concept of bioactive cell-penetrating peptides.

Cell-penetrating peptides (CPPs) have proven utility for the highly efficient intracellular delivery of bioactive cargoes that include peptides, proteins, and oligonucleotides. The many strategies developed to utilize CPPs solely as pharmacokinetic modifiers necessarily requires them to be relatively inert. Moreover, it is feasible to combine one or multiple CPPs with bioactive cargoes either by direct chemical conjugation or, more rarely, as non-covalent complexes.

Influence of stearyl and trifluoromethylquinoline modifications of the cell penetrating peptide TP10 on its interaction with a lipid membrane.

The PepFect family of cell-penetrating peptides (CPPs) was designed to improve the delivery of nucleic acids across plasma membranes. We present here a comparative study of two members of the family, PepFect3 (PF3) and PepFect6 (PF6), together with their parental CPP transportan-10 (TP10), and their interactions with lipid membranes. We show that the addition of a stearyl moiety to TP10 increases the amphipathicity of these molecules and their ability to insert into a lipid monolayer composed of zwitterionic phospholipids.

Cholesterol prevents interaction of the cell-penetrating peptide transportan with model lipid membranes.

Interaction of the cell-penetrating peptide (CPP) cysteine-transportan (Cys-TP) with model lipid membranes was examined by spin-label electron paramagnetic resonance (EPR). Membranes were labeled with lipophilic spin probes and the influence of Cys-TP on membrane structure was studied. The influence of Cys-TP on membrane permeability was monitored by the reduction of a liposome-trapped water-soluble spin probe.

Role of cysteine 341 and arginine 348 of GLP-1 receptor in G-protein coupling.

We have demonstrated the ability of peptides derived from the third intracellular loop of GLP-1 receptor to differently modulate activity of four different types of G-proteins overexpressed in sf9 cells. In this respect, the involvement of Cys(341) in inhibition of G(s) and Cys(341) in activation of G(s) and in inhibition of G(i1,) G(o), and G(11), respectively, indicates their potential role in discrimination between different types of G-proteins. Moreover, these two amino acids from the third intracellular loop might represent an important novel targets for covalent modification by downstream regulators in signaling through GLP-1 receptor.

Histamine release, an undesired effect of thrombin inhibitors with basic character, is mediated through direct activation of G(i) proteins.

The common structural feature of LK direct thrombin inhibitors is a strong basic group attached to the azaphenylalanine scaffold, which is important for the appropriate interaction at the thrombin active site. Our previous results have shown that this basic group could be responsible for a reduction of tracheal air flow and a fall of mean arterial pressure in anaesthetized rats, an undesired effect of direct thrombin inhibitors which correlated with their ability to release histamine from mast cells. In the present study, we investigated the mechanism of LK direct thrombin inhibitors-induced histamine release from rat peritoneal mast cells.

Distribution of organochlorine pollutants in ovine dental tissues and bone.

The distribution of selected lipophilic organochlorine pollutants, including two pairs of tetra- and hexa-chlorobiphenyl isomers (PCB-54, -80, -155, -169) and organochlorine pesticides [hexachlorobenzene (HCB) and 1,1-bis(4-chlorophenyl)-2,2-dichloroethene (4,4'-DDE)], in ovine dental pulp, dentine, enamel and mandibular bone was examined. Sheeps were given a single dose of individual organochlorine (1-4μmol/kg) in olive oil by intramuscular injection and sacrificed 2 months later. Organochlorine residues were determined by gas chromatography.

Multiple interaction sites of galnon trigger its biological effects.

Galnon was first reported as a low molecular weight non-peptide agonist at galanin receptors [Saar et al. (2002) Proc. Natl.

Inhibition of erythrocyte acetylcholinesterase by n-butanol at high concentrations.

Erythrocyte acetylcholinesterase (AChE) is bound to the membrane by a complex glycosylphosphatidylinositol anchor, so the effect of alcohol on AChE activity may reflect direct and/or membrane-mediated effects. The indication of a direct interaction between n-butanol and AChE molecules is the activation/inhibition of AChE by occupation of the enzyme's active and/or regulatory sites by alcohol. The activation of AChE can occur only at low concentrations of alcohols, while at high concentrations AChE is inhibited.

Cell-penetrating peptides: mechanism and kinetics of cargo delivery.

Cell-penetrating peptides (CPPs) are short peptides of less than 30 amino acids that are able to penetrate cell membranes and translocate different cargoes into cells. The only common feature of these peptides appears to be that they are amphipathic and net positively charged. The mechanism of cell translocation is not known but it is apparently receptor and energy independent although, in certain cases, translocation can be partially mediated by endocytosis.

The membrane lateral domain approach in the studies of lipid-protein interaction of GPI-anchored bovine erythrocyte acetylcholinesterase.

A novel membrane lateral domain approach was used to test whether the activity of the membrane-bound enzyme acetylcholinesterase (AChE) depends on the local properties (e.g. local lipid ordering) of bovine erythrocyte-ghost membrane.

Molecular characterization of specific H1-receptor agonists histaprodifen and its Nalpha-substituted analogues on bovine aortic H1-receptors.

We determined the molecular properties of the selective and potent H(1)-receptor agonist histaprodifen and its N(alpha) substituted analogues: methyl-, dimethyl-, and imidazolylethyl-histaprodifen (suprahistaprodifen). All derivatives show high affinity for (3)H-mepyramine labeled bovine aortic H(1)-receptor binding sites with the following order of potency: suprahistaprodifen > dimethylhistaprodifen > methylhistaprodifen > histaprodifen > histamine. Suprahistaprodifen and dimethylhistaprodifen were the most potent displacers of (3)H-mepyramine binding (K(i)=4.

Different role of intracellular loops of glucagon-like peptide-1 receptor in G-protein coupling.

Previous studies revealed the importance of the third intracellular loop of glucagon-like peptide-1 receptor (GLP-1R) in coupling to G(s) and G(i1) proteins. In order to further study the signaling mechanisms of GLP-1R, we tested three peptides, corresponding to the sequences of the first (IC(1)), the second (IC(2)), and the third (IC(3)) intracellular loop of GLP-1R, for their interactions with heterotrimeric G-proteins of different types (G(alphas), G(alphao), G(alphai1), and G(alpha11) plus G(beta1gamma2)) overexpressed in sf9 cells. IC(3) peptide powerfully stimulates all types of tested G-proteins, whereas IC(1) and IC(2) peptides show differential effects on G-proteins.

Membrane-bound progesterone receptors coupled to G proteins in the fungus Rhizopus nigricans.

Steroid binding sites with high affinity for progesterone (Kd=40+/-14 nM determined by binding, and Kd=71+/-22 nM determined by displacement studies) and lower affinity for 21-hydroxyprogesterone and for testosterone, but no affinity for estradiol-17beta, onapristone and alpha-naphthoflavone were detected in the enriched plasma membrane fraction of the fungus Rhizopus nigricans. The amount of steroid binding sites is in accordance with the value of B(max)=744+/-151 fmol (mg protein)(-1). In the membrane fraction, progesterone induced about 30% activation of G proteins over basal level, as determined by GTPase activity (EC50=32+/-8 nM) and by the guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) binding rate (EC50=61+/-21 nM).

G-Protein coupled progesterone receptors in the plasma membrane of fungus Rhizopus nigricans.

We have demonstrated simultaneous existence of progesterone receptors and GTPase activity in the membranes prepared from the filamentous fungus Rhizopus nigricans. The results obtained with pertussis toxin treated fungal mycelium suggest that these receptors do not couple to G-G-proteins and play a role in the induction of steroid hydroxylating enzyme system by steroid substrates in the fungus.