Farnesylation-defective Rheb Increases Axonal Length Independently of mTORC1 Activity in Embryonic Primary Neurons

Rheb (Ras homolog enriched in the brain) is a small GTPase protein that plays an important role in cell signaling for development of the neocortex through modulation of mTORC1 (mammalian-target-of-rapamycin-complex-1) activity. mTORC1 is known to control various biological processes including axonal growth in forming complexes at the lysosomal membrane compartment. As such, anchoring of Rheb on the lysosomal membrane via the farnesylation of Rheb at its cysteine residue (C180) is required for its promotion of mTOR activity. To test the significance of Rheb farnesylation, we overexpressed a farnesylation mutant form of Rheb, Rheb C180S, in primary rat hippocampal neurons and also in mouse embryonic neurons using in utero electroporation. Interestingly, we found that Rheb C180S maintained promotional effect of axonal elongation similar to the wild-type Rheb in both test systems. On the other hand, Rheb C180S failed to exhibit the multiple axon-promoting effect which is found in wild-type Rheb. The levels of phospho-4EBP1, a downstream target of mTORC1, were surprisingly increased in Rheb C180S transfected neurons, despite the levels of phosphorylated mTOR being significantly decreased compared to control vector transfectants. A specific mTORC1 inhibitor, rapamycin, also could not completely abolish axon elongation characteristics of Rheb C180S in transfected cells. Our data suggests that Rheb in a non-membrane compartment can promote the axonal elongation via phosphorylation of 4EBP1 and through an mTORC1-independent pathway.

Interacciones moleculares de un flavonoide prenilado con transportadores de antimicóticos dependientes de ATP / Molecular interactions of a prenylated flavonoid with ATP-depending antimycotic transporters

Uno de los principales mecanismos para la resistencia de Candida albicans a azoles es la sobreexpresión de transportadores que extruyen los fármacos antimicóticos y disminuyen su concentración intracelular. Anteriormente nuestros laboratorios habían informado que el flavonoide prenilado 2', 4'-dihidroxi - 5'-(1'''-dimetilalil)-6-prenil pinocembrina (6PP) inhibe en forma competitiva los transportadores cdr, dependientes del ATP, además de ser antimicrobiano per se. En este trabajo realizamos estudios bioinformáticos para analizar los posibles sitios de interacción molecular estereoespecífica del 6PP, fluconazol y adenosín trifosfato (ATP) con los transportadores cdr1 y cdr2. Se confeccionaron los modelos tridimensionales de los ligandos y de los transportadores a través de estudios de homología de datos respecto de otras macromoléculas. Se calculó la variación de energía libre de Gibbs ( G) asociada a 45 modelos de interacción molecular y se estudiaron cdr1 y cdr2 en formacomparativa, dada su similitud estructural. Se seleccionaron los modelos más probables desde el punto de vista termodinámico. El 6PP y el fluconazol podrían competir de diversas maneras por un sitio estereoespecífico que atraviesa la membrana de la levadura. Este sitio, numerado como 1, fue estudiado exhaustivamente, es común a los transportadores cdr1 y cdr2 y aporta selectivamente distintosaminoácidos. Entre estos últimos, los de mayor participación en los modelos de interacción fueron, en cdr1 y cdr2, respectivamente: PHE 754,756 y TYR 747,749 . Se seleccionaron los 9 mejores complejos formados por cada ligando, de acuerdo a su energía de afinidad decreciente (en KJ/mol en mejor modelo de cdr2: 6PP = -9,3; fluconazol = -6,8). Además el flavonoide prenilado competiría con el ATP en el dominio citosólico y con menor probabilidad lo imitaría el fluconazol ( G en KJ/mol en mejor modelo de cdr2: 6P P= -8,2, ATP = -7,6; fluconazol = -6,7)...

Isoprenoid biosynthesis in the erythrocytic stages of Plasmodium falciparum

The development of new drugs is one strategy for malaria control. Biochemical pathways localised in the apicoplast of the parasite, such as the synthesis of isoprenic precursors, are excellent targets because they are different or absent in the human host. Isoprenoids are a large and highly diverse group of natural products with many functions and their synthesis is essential for the parasite's survival. During the last few years, the genes, enzymes, intermediates and mechanisms of this biosynthetic route have been elucidated. In this review, we comment on some aspects of the methylerythritol phosphate pathway and discuss the presence of diverse isoprenic products such as dolichol, ubiquinone, carotenoids, menaquinone and isoprenylated proteins, which are biosynthesised during the intraerythrocytic stages of Plasmodium falciparum.