Synthesis of functionalized rab GTPases by a combination of solution- or solid-phase lipopeptide synthesis with expressed protein ligation

Prenylated proteins with non-native functionalities are generally very difficult to obtain by recombinant or enzymatic means. The semisynthesis of preparative amounts of prenylated Rab guanosine triphosphatases (GTPases) from recombinant proteins and synthetic prenylated peptides depends largely on the availability of functionalised prenylated peptides corresponding to the proteins' native structure or modifications thereof. Here, we describe and compare solution-phase and solid-phase strategies for the generation of peptides corresponding to the prenylated C terminus of Rab7 GTPase. The solid-phase with utilisation of a hydrazide linker emerges as the more favourable approach. It allows a fast and practical synthesis of pure peptides and gives a high degree of flexibility in their modification. To facilitate the analysis of semisynthetic proteins, the synthesised peptides were equipped with a fluorescent group. Using the described approach, we introduced fluorophores at several different positions of the Rab7 C terminus. The position of the incorporated fluorescent groups in the peptides did not influence the protein-ligation reaction, as the generated peptides could be ligated onto thioester-tagged Rab7. However, it was found that the positioning of the fluorescent group had an influence on the functionality of the Rab7 proteins; analysis of the interaction of the semisynthetic Rab7 proteins with REP (Rab escort protein) and GDI (guanosine diphosphate dissociation inhibitor) molecules revealed that modification of the peptide side chains or of the C-terminal isoprenoid did not significantly interfere with complex formation. However, functionalisation of the C terminus was found to have an adverse effect on complex formation and stability, possibly reflecting low structural flexibility of the Rab GDI/REP molecules in the vicinity of the lipid-binding site.     

Solid-phase synthesis of lipidated peptides

A new flexible and efficient methodology for the solid-phase synthesis of lipidated peptides has been developed. The approach is based on the use of previously synthesized building blocks and overcomes the limitations of previously reported methods, since long doubly lipidated peptides can be synthesized by using this route. Furthermore, it was thus possible to prepare a large number of N- and H-Ras peptides bearing a wide range of reporter and/or linking groups--efficient tools for the investigation of biological processes. In terms of efficiency and flexibility this solid-phase method is superior to the solution-phase synthesis. It gives pure peptides in multimilligram amounts within a much shorter time and with superior overall yield.     

Analysis of protective action of ischemic preconditioning from fluorescence spectra of blood serum and probes added to it

The protective effect of ischemic preconditioning on brain tissue during total ischemia is shown by using spectral-luminescence methods for estimating oxidative damage to blood components after ischemia. Fluorescence of blood serum and molecular probes added to it is investigated after producing brain ischemia with preconditioning. Lipid free-radical peroxide damage of low-density lipoproteins is found to decrease after producing ischemia with preconditioning rather than ischemia without preconditioning. __________ Translated from Zhurnal Prikladnoi Spektroskopii Vol. 74, No. 3, pp. 378–381, May–June, 2007.     

Open tubular capillary electrochromatography: A useful microreactor for collagen I glycation and interaction studies with low-density lipoprotein particles

Diabetes, a multifunctional disease and a major cause of morbidity and mortality in the industrialized countries, strongly associates with the development and progression of atherosclerosis. One of the consequences of high level of glucose in the blood circulation is glycation of long-lived proteins, such as collagen I, the most abundant component of the extracellular matrix (ECM) in the arterial wall. Glycation is a long-lasting process that involves the reaction between a carbonyl group of the sugar and an amino group of the protein, usually a lysine residue. This reaction generates an Amadori product that may evolve in advanced glycation end products (AGEs). AGEs, as reactive molecules, can provoke cross-linking of collagen I fibrils. Since binding of low-density lipoproteins (LDLs) to the ECM of the inner layer of the arterial wall, the intima, has been implicated to be involved in the onset of the development of an atherosclerotic plaque, collagen modifications, which can affect the affinity of native and oxidized LDL for collagen I, can promote the entrapment of LDLs in the intima and accelerate the progression of atherosclerosis.In this study, open tubular capillary electrochromatography is proposed as a new microreactor to study in situ glycation of collagen I. The kinetics of glycation was first investigated in a fused silica collagen I-coated capillary. Dimethyl sulphoxide, injected as an electroosmotic flow marker, gave information about the charge of coating. Native and oxidized LDL, and selected peptide fragments from apolipoprotein B-100, the protein covering LDL particles, were injected as marker compounds to clarify the interactions between LDLs and the glycated collagen I coating. The method proposed is simple and inexpensive, since only small amounts of collagen and LDL are required. Atomic force microscopy images complemented our studies, highlighting the difference between unmodified and glycated collagen I surfaces.     

MRI-guided neutron capture therapy by use of a dual gadolinium/boron agent targeted at tumour cells through upregulated low-density lipoprotein transporters

The upregulation of low-density lipoprotein (LDL) transporters in tumour cells has been exploited to deliver a sufficient amount of gadolinium/boron/ligand (Gd/B/L) probes for neutron capture therapy, a binary chemio-radiotherapy for cancer treatment. The Gd/B/L probe consists of a carborane unit (ten B atoms) bearing an aliphatic chain on one side (to bind LDL particles), and a Gd(III)/1,4,7,10-tetraazacyclododecane monoamide complex on the other (for detection by magnetic resonance imaging (MRI)). Up to 190 Gd/B/L probes were loaded per LDL particle. The uptake from tumour cells was initially assessed on cell cultures of human hepatoma (HepG2), murine melanoma (B16), and human glioblastoma (U87). The MRI assessment of the amount of Gd/B/L taken up by tumour cells was validated by inductively coupled plasma-mass-spectrometric measurements of the Gd and B content. Measurements were undertaken in vivo on mice bearing tumours in which B16 tumour cells were inoculated at the base of the neck. From the acquisition of magnetic resonance images, it was established that after 4-6 hours from the administration of the Gd/B/L-LDL particles (0.1 and 1 mmol kg(-1) of Gd and (10)B, respectively) the amount of boron taken up in the tumour region is above the threshold required for successful NCT treatment. After neutron irradiation, tumour growth was followed for 20 days by MRI. The group of treated mice showed markedly lower tumour growth with respect to the control group.     

Action de l'AMP-c sur les monocouches mixtes de céphaéline-albumine d'œuf

Résumé En vue de vérifier si l'AMP-cyclique produit une action expansive lorsqu'il agit sur les monocouches de lipoprotéines mixtes ces monocouches insolubles ont été préparées avec de la céphaéline et de l'albumine dóeuf.Ces monocouches ont montré une bonne miscibilité entre les deux composants, et seules de faibles actions réciproques ont été observées.La pénétration de l'AMP-cyclique dans ces monocouches mixtes produit un effet expansif appréciable, son action atteignant le niveau maximal lorsque la concentration de l'AMP-cyclique dans la sous-phase est de 10^–6 M (concentration normale de la cellule).Un effet similaire sur les membranes mixtes de lipoprotéines de la paroi cellulaire est attribué a l'AMP-cyclique.

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With a view to checking whether cyclic-AMP produces an expansive action when acting on mixed lipoprotein monolayers, such insoluble monolayers were prepared with cephalin and egg albumin. These layers showed a good miscibility between the two components, with only weak interactions being observed.The penetration of cyclic-AMP in these mixed monolayers produces an appreciable expansive effect, its action reaching a maximum level when the cyclic-AMP concentration in the subphase is 10^–6 M (standard cell concentration).A similar effect on the lipoprotein mixed membranes of the cell wall is attributed to cyclic-AMP.

Directeur du Département: Prof. Dr. Serafín Garcia Fernández     

Lipidated ras and rab peptides and proteins--synthesis, structure, and function

Chemical biology can be defined as the study of biological phenomena from a chemical approach. Based on the analysis of relevant biological phenomena and their structural foundation, unsolved problems are identified and tackled through a combination of chemistry and biology. Thus, new synthetic methods and strategies are developed and employed for the construction of compounds that are used to investigate biological procedures. Solid-phase synthesis has emerged as the preferred method for the synthesis of lipidated peptides, which can be chemoselectively ligated to proteins of the Ras superfamily. The generated peptides and proteins have solved biological questions in the field of the Ras-superfamily GTPases that are not amendable to chemical or biological techniques alone.     

Multipronged Biomimetic Approach To Create Optically Tunable Nanoparticles

Current biomimetics for medical applications use a single biomimetic approach to imitate natural structures, which can be insufficient for reconstructing structurally complex natural systems. Multipronged efforts may resolve these complexities. To achieve interesting nanostructure‐driven optical properties, a dual‐biomimetic system contained within a single nanoagent was engineered to recapitulate chlorosomes, efficient light‐harvesting organelles that have unique dye assemblies and tunable photonic properties. A series of chlorin dyes was synthesized, and these hydrophobic assemblies were stabilized inside a high‐density lipoprotein, a second biomimetic that enabled in vivo utility. This system resulted in tunable tumor imaging of intact (photoacoustic) and disrupted (activatable fluorescence) nanostructures. The successful demonstration of this multipronged biomimetic approach opens the door for reconstruction of complex natural systems for biomedical applications.