A unique chaperoning mechanism in class A JDPs recognizes and stabilizes mutant p53.

J-domain proteins (JDPs) constitute a large family of molecular chaperones that bind a broad spectrum of substrates, targeting them to Hsp70, thus determining the specificity of and activating the entire chaperone functional cycle. The malfunction of JDPs is therefore inextricably linked to myriad human disorders. Here, we uncover a unique mechanism by which chaperones recognize misfolded clients, present in human class A JDPs. Through a newly identified ß-hairpin site, these chaperones detect changes in protein dynamics at the initial stages of misfolding, prior to exposure of hydrophobic regions or large structural rearrangements. The JDPs then sequester misfolding-prone proteins into large oligomeric assemblies, protecting them from aggregation. Through this mechanism, class A JDPs bind destabilized p53 mutants, preventing clearance of these oncoproteins by Hsp70-mediated degradation, thus promoting cancer progression. Removal of the ß-hairpin abrogates this protective activity while minimally affecting other chaperoning functions. This suggests the class A JDP ß-hairpin as a highly specific target for cancer therapeutics.

Unveiling the membrane bound dihydroorotate: Quinone oxidoreductase from Staphylococcus aureus.

Staphylococcus aureus is an opportunistic pathogen and one of the most frequent causes for community acquired and nosocomial bacterial infections. Even so, its energy metabolism is still under explored and its respiratory enzymes have been vastly overlooked. In this work, we unveil the dihydroorotate:quinone oxidoreductase (DHOQO) from S. aureus, the first example of a DHOQO from a Gram-positive organism. This protein was shown to be a FMN containing menaquinone reducing enzyme, presenting a Michaelis-Menten behaviour towards the two substrates, which was inhibited by Brequinar, Leflunomide, Lapachol, HQNO, Atovaquone and TFFA with different degrees of effectiveness. Deletion of the DHOQO coding gene (Δdhoqo) led to lower bacterial growth rates, and effected in cell morphology and metabolism, most importantly in the pyrimidine biosynthesis, here systematized for S. aureus MW2 for the first time. This work unveils the existence of a functional DHOQO in the respiratory chain of the pathogenic bacterium S. aureus, enlarging the understanding of its energy metabolism.

Stabilization of a DNA aptamer by ligand binding.

G-rich aptamers such as AS1411 are small oligonucleotides that present several benefits comparatively to monoclonal antibodies, since they are easier to manufacture and store, have small size and do not stimulate an immune response. We analyzed AT11-B1, a modified sequence of AT11 (itself a modified version of AS1411), in which one thymine was removed from the bulge region. We studied G-quadruplex (G4) formation/stabilization using PhenDC3, PDS, BRACO-19, TMPyP4 and 360A ligands by different biophysical techniques, namely circular dichroism (CD), Förster resonance energy transfer (FRET-melting) and nuclear magnetic resonance (NMR). The CD spectra showed that AT11-B1 adopts a predominant G4 of parallel topology when the buffer contains KCl or when ligands are added. PhenDC3 induced a ΔTm of 30 °C or more of the G4 structure as shown by CD- and FRET-melting experiments. The ligands demonstrate high affinity for AT11-B1 G4 and the NMR studies revealed that the AT11-B1 G4 involves four G-tetrad layers. The in silico studies suggest that all ligands bind AT11-B1 G4, namely, by stacking interactions, with the possible exception of PDS that may bind to the loop/groove interface. In addition, molecular dynamics simulations revealed that nucleolin (NCL) interacts with the AT11-B1 G4 structure through the RNA binding domain (RBD) 2 and the 12-residue linker between RBD1,2. Moreover, AT11-B1 G4 was internalized into a NCL-positive tongue squamous cell carcinoma cell line. In a nutshell, this study may help the identification of the ligands scaffolds to bind and stabilize AT11-B1, improving the targeting towards NCL that is overexpressed in cancer cells.