Asymmetric post-translational modifications regulate the nuclear translocation of STAT3 homodimers in response to leukemia inhibitory factor.

STAT3 is a pleiotropic transcription factor overactivated in 70% of solid tumours. We have recently reported that inactivating mutations on residues susceptible to post-translational modifications (PTMs) in only one of the monomers (i.e. asymmetric) caused changes in the cellular distribution of STAT3 homodimers. Here, we used more controlled experimental conditions, i.e. without the interference of endogenous STAT3 (STAT3-/- HeLa cells) and in the presence of a defined cytokine stimulus (Leukemia Inhibitory Factor, LIF), to provide further evidence that asymmetric PTMs affect the nuclear translocation of STAT3 homodimers. Time-lapse microscopy for 20 min after LIF stimulation showed that S727 dephosphorylation (S727A) and K685 inactivation (K685R) slightly enhanced the nuclear translocation of STAT3 homodimers, while K49 inactivation (K49R) delayed STAT3 nuclear translocation. Our findings suggest that asymmetrically modified STAT3 homodimers could be a new level of STAT3 regulation and, therefore, a potential target for cancer therapy.

Synthesis, Base Pairing Properties, and Biological Activity Studies of Platinum(II) Complexes Based on Uracil Nucleosides.

The synthesis and base pairing properties of platinum complexes based on uridine and deoxyuridine nucleosides and preliminary studies of their antiproliferative activity are described. Platinum(II) uridine and deoxyuridine complexes were synthesized by C-I oxidative addition to Pt(0)(PPh3)4. First, the synthesis was performed with protected nucleosides to generate complexes 1 and 2, which were deprotected under basic conditions, affording complexes 3 and 4 in good yields. The synthesis with the unprotected nucleosides was also performed and provided complexes 3 and 4 effectively. Base pairing interactions were measured for complex 1, either for self-base pairing or for the Watson-Crick base pair. Complex 1 undergoes self-base pairing in CDCl3, and this aggregation was found not to be dependent on metalation. Contrastingly, for the Watson-Crick base pair with adenine, base pairing was also observed, but metalation was found to affect hydrogen bonding considerably. Complexes 3 and 4 and the corresponding ligand precursors were evaluated for their antiproliferative activity against human glioblastoma cell line U-251. The compounds showed IC50 values of 3.30 (3) and 1.84 (4) µM but are also toxic for nontumorous cell lines.

Sacsin Deletion Induces Aggregation of Glial Intermediate Filaments.

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a neurodegenerative disorder commonly diagnosed in infants and characterized by progressive cerebellar ataxia, spasticity, motor sensory neuropathy and axonal demyelination. ARSACS is caused by mutations in the SACS gene that lead to truncated or defective forms of the 520 kDa multidomain protein, sacsin. Sacsin function is exclusively studied on neuronal cells, where it regulates mitochondrial network organization and facilitates the normal polymerization of neuronal intermediate filaments (i.e., neurofilaments and vimentin). Here, we show that sacsin is also highly expressed in astrocytes, C6 rat glioma cells and N9 mouse microglia. Sacsin knockout in C6 cells (C6Sacs-/-) induced the accumulation of the glial intermediate filaments glial fibrillary acidic protein (GFAP), nestin and vimentin in the juxtanuclear area, and a concomitant depletion of mitochondria. C6Sacs-/- cells showed impaired responses to oxidative challenges (Rotenone) and inflammatory stimuli (Interleukin-6). GFAP aggregation is also associated with other neurodegenerative conditions diagnosed in infants, such as Alexander disease or Giant Axonal Neuropathy. Our results, and the similarities between these disorders, reinforce the possible connection between ARSACS and intermediate filament-associated diseases and point to a potential role of glia in ARSACS pathology.

Can asymmetric post-translational modifications regulate the behavior of STAT3 homodimers?

Signal transducer and activator of transcription 3 (STAT3) is a ubiquitous and pleiotropic transcription factor that plays essential roles in normal development, immunity, response to tissue damage and cancer. We have developed a Venus-STAT3 bimolecular fluorescence complementation assay that allows the visualization and study of STAT3 dimerization and protein-protein interactions in living cells. Inactivating mutations on residues susceptible to post-translational modifications (PTMs) (K49R, K140R, K685R, Y705F and S727A) changed significantly the intracellular distribution of unstimulated STAT3 dimers when the dimers were formed by STAT3 molecules that carried different mutations (ie they were "asymmetric"). Some of these asymmetric dimers changed the proliferation rate of HeLa cells. Our results indicate that asymmetric PTMs on STAT3 dimers could constitute a new level of regulation of STAT3 signaling. We put forward these observations as a working hypothesis, since confirming the existence of asymmetric STAT3 homodimers in nature is extremely difficult, and our own experimental setup has technical limitations that we discuss. However, if our hypothesis is confirmed, its conceptual implications go far beyond STAT3, and could advance our understanding and control of signaling pathways.

Synthesis and antimalarial evaluation of prodrugs of novel fosmidomycin analogues.

The continuous development of drug resistance by Plasmodium falciparum, the agent responsible for the most severe forms of malaria, creates the need for the development of novel drugs to fight this disease. Fosmidomycin is an effective antimalarial and potent antibiotic, known to act by inhibiting the enzyme 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR), essential for the synthesis of isoprenoids in eubacteria and plasmodia, but not in humans. In this study, novel constrained cyclic prodrug analogues of fosmidomycin were synthesized. One, in which the hydroxamate function is incorporated into a six-membered ring, was found have higher antimalarial activity than fosmidomycin against the chloroquine and mefloquine resistant P. falciparum Dd2 strain. In addition, it showed very low cytotoxicity against cultured human cells.