Matjaž Zorko

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. Both IC(1) and IC(2) peptides activate G(s) and cooperate with IC(3) peptide in its stimulation. G(o) is not affected by IC(1) and IC(2). G(i1) and G(11) are not affected by IC(1), but are activated by IC(2), which in activation cooperates with IC(3). We suggest that GLP-1R is not coupled only to G(s) and G(i1), as shown previously, but also to G(o) and G(11). IC(3) loop is the main switch that mediates signaling via GLP-1R to G-proteins, while IC(1) and IC(2) loops are important in discrimination between different types of G-proteins.

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. Acad. Sci. USA 99, 7136-7141]. Following its systemic administration, this synthetic ligand affected a range of important physiological processes including appetite, seizures and pain. Physiological activity of galnon could not be explained solely by the activation of the three known galanin receptors, GalR1, GalR2 and GalR3. Consequently, it was possible that galnon generates its manifold effects by interacting with other signaling pathway components, in addition to via GalR1-3. In this report, we establish that galnon: (i) can penetrate across the plasma membrane of cells, (ii) can activate intracellular G-proteins directly independent of receptor activation thereby triggering downstream signaling, (iii) demonstrates selectivity for different G-proteins, and (iiii) is a ligand to other G-protein coupled receptors (GPCRs) in addition to via GalR1-3. We conclude that galnon has multiple sites of interaction within the GPCR signaling cascade which mediate its physiological effects.

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. The addition of negatively charged phospholipids into the monolayer results in decreased binding and insertion of the stearylated peptides, indicating modification in the balance of hydrophobic versus electrostatic interactions of peptides with lipid bilayer, thus revealing some clues for the selective interaction of these CPPs with different lipids. The trifluoromethylquinoline moieties, in PF6 make no significant contribution to membrane binding and insertion. TP10 actively introduces pores into the bilayers of large and giant unilamellar vesicles, while PF3 and PF6 do so only at higher concentrations. This is consistent with the lower toxicity of PF3 and PF6 observed in previous studies.

Kinetic characterization of all steps of the interaction between acetylcholinesterase and eserine

The three-step carbamylenzyme mechanism of the action of eserine on acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) has been known for a long time, but its complete kinetic characterization has never been done. Some of our investigations indicated that the determination of missing kinetic parameters should include the inspection of the enzyme-eserine interaction in a very wide range of eserine concentrations. Therefore, the activity of acetylcholinesterase as a function of time in the presence of low concentrations of eserine comparable to the enzyme concentration was followed. The reaction mechanism was analysed by fitting numerically integrated differential equations that describe the time dependences of all reactants and reaction intermediates to these data. Additionally, the progress curve measurements at higher eserine concentrations were carried out on a stopped-flow apparatus. The corresponding progress curve equations were derived and the kinetic parameters evaluated by non-linear regression treatment. The complex analysis confirmed the three-step mechanism. The values of the constants showed that the very high affinity of eserine for binding into the active centre of the enzyme is not so much a consequence of the fast initial complex formation but rather a consequence of its slow dissociation. The subsequent covalent bonding of eserine is also slow, but faster than the dissociation of the initial complex. In this manner, the decarbamoylation is the only process responsible for the reactivation of acetylcholinesterase after removal of eserine.

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. This issue has an additional aspect of interest due to an alternative mode of insertion of AChE molecules into the membrane by the glycosylphosphatidylinositol (GPI) anchor. In our experiments the lateral domain membrane structure was influenced by temperature and by the addition of n-butanol, and was quantitatively characterized using the method of EPR spectrum decomposition. The activity of AChE was determined by a colorimetric assay in the same samples. The results show that the membrane stabilizes the conformation of the membrane-bound AChE compared to the isolated AChE. In addition, a correlation was observed between the temperature dependence of order parameter of the most-ordered domain type and the activity of AChE. Therefore, our findings support the idea that the function of GPI proteins can be modulated by the lipid bilayer. Based on the assumption that the overall activity of AChE depends on the order parameters of particular domain types as well as their proportions, two models for AChE activity were introduced. In the first, a random distribution of enzyme molecules was proposed, and in the second, localization of enzyme molecules in a single (cholesterol-rich) domain type was assumed. Better agreement between measured and calculated activity values speaks in favor of the second model.

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. Cys‐TP caused lipid ordering in membranes prepared from pure dimyristoylphosphatidylcholine (DMPC) and in DMPC membranes with moderate cholesterol concentration. In addition, Cys‐TP caused a large increase in permeation of DMPC membranes. In contrast, with high cholesterol content, at which model lipid membranes are in the so‐called liquid‐ordered phase, no effect of Cys‐TP was observed, either on the membrane structure or on the membrane permeability. The interaction between Cys‐TP and the lipid membrane therefore depends on the lipid phase. This could be of great importance for understanding of the CPP–lipid interaction in laterally heterogeneous membranes, while it implies that the CPP–lipid interaction can be different at different points along the membrane. Copyright

The acceleration of methanesulfonylation of acetylcholinesterase with cationic accelerators as an electrostatic effect.

1. In order to check our hypothesis of the electrostatic nature of the acceleration of methanesulfonylation of acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) with cationic accelerators, equations were solved for methane-sulfonylation with two accelerators and the reaction was studied in the presence of some single accelerators, including the sodium cation, and in the presence of two acclerators simultaneously. 2. The second-order rate constants for methanesulfonylation of the complexes between the enzyme and accelerators decamethonium, tetraethylammonium and tetramethylammonium are 90, 88 and 17 1 - mol-1 - s-1, respectively, which corresponds to a maximal acceleration of 29, 28 and 5.5 times, respectively. The dissociation constants for the binding of these accelerators to the enzyme, obtained from our acceleration experiments, are 3.7 - 10(-6), 3.2 - 10(-4) and 1.4 - 10(-3) M, respectively. These values are in good agreement with the dissociation constants of these ligands as inhibitors of acetylcholinesterase. It is interesting to note that the sodium cation also accelerates the methane-sulfonylation up to around three times, the corresponding second-order rate constant and the dissociation constant being 10 1 - mol-1 - s-1 and 1.3 M, respectively. 3. All tested cations compete in the acceleration with each other; they seem to accelerate the reaction in the same way and from the same site, the catalytic anionic site. 4. These findings confirm the hypothesis of the electrostatic nature of acceleration.

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 Cys341 in inhibition of Gs and Cys341 in activation of Gs and in inhibition of Gi1, Go, and G11, 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.

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

Abstract 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 Gi-Go-proteins and play a role in the induction of steroid hydroxylating enzyme system by steroid substrates in the fungus.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 Gi-Go-proteins and play a role in the induction of steroid hydroxylating enzyme system by steroid substrates in the fungus.