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.

Structural Perspective into the Interaction of an Oncogenesis-Relevant pre-miRNA G-Quadruplex Ligand Carrier with the Protein Nucleolin.

The structural determinants of the interaction of the G-quadruplex (G4) motif found in precursor miRNA 149 (rG4) with the acridine orange derivative C8 , a G4 ligand stabilizer possessing anticancer activity, and the protein nucleolin (overexpressed in cancer cells) were investigated by Nuclear Magnetic Resonance (NMR) spectroscopy. For the rG4/C8 complex, the results revealed a strong stabilizing interaction between the aromatic core and the iodinated ring of the C8 ligand with the rG4 structure. The NMR study revealed also different interaction patterns between nucleolin and rG4 and nucleolin and rG4/C8 complex. In the absence of the ligand, rG4 establishes interactions with polar residues of the protein while for the rG4/C8 complex, these contacts are mainly established with amino acids that have hydrophobic side chains. However, nucleolin chemical shift perturbation studies in the presence of rG4 or rG4/C8 reveal the same location between domains 1 and 2 of the protein, which suggests that the rG4 and rG4/C8 complex bind in this region. This puzzling structural study opens a new framework to study rG4/ligand/nucleolin complexes that might impact the biogenesis of miRNA 149.

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.

Instrumentação reciprocante em canal mésiovestibular de molar superior após fratura de lima endodôntica: relato de caso / Reciprocal instrumentation in the mesiobuccal canal of the upper molar after endodontic file fracture: case report

Objetivo: reportar um caso clínico de ultrapassagem de instrumento endodôntico fraturado em terço médio de canal mésiovestibular de molar superior permanente, utilizando a técnica de instrumentação reciprocante associada à instrumentação convencional manual e ao gel de E.D.T.A. 24%. Relato de caso: paciente do sexo masculino, 24 anos, normossistêmico, compareceu ao consultório odontológico privado com queixa de dor espontânea e encaminhamento para tratamento endodôntico do dente 17. O diagnóstico foi pulpite irreversível sintomática com indicação de tratamento endodôntico primário. A lima principal escolhida foi a Reciproc® R25 M-wire (VDW), que, durante a instrumentação do canal mésiovestibular, sofreu fratura por torção em terço médio, inviabilizando a continuação em direção apical. Considerações finais: a associação da técnica convencional manual das limas Hedströen com a técnica reciprocante de corte reverso da lima Wave One® Gold Primary e o uso do gel de E.D.T.A. 24% permitiram ultrapassagem do fragmento de lima fraturado em terço médio do canal mésiovestibular do dente 17, promovendo selamento hermético, limpeza, modelagem e obturação satisfatórias.(AU)

Objective: to report a clinical case of a fractured endodontic instrument in the middle third of the permanent maxillary molar maxillary canal using the technique instrumentation reciprocation with technique to conventional manual instrumentation and the E.D.T.A. 24% gel. Case report: a 24-yearold normosystemic male patient came to the private dental office complaining of spontaneous pain and referred for endodontic treatment of tooth 17. The diagnosis was irreversible symptomatic pulpitis with indication for primary endodontic treatment. The main file chosen was the Reciproc® R25 M-wire (VDW), which during the instrumentation of the mesiobuccal canal suffered torsional fracture in the middle third, preventing continuity in the apical direction. Final considerations: the association of the conventional manual Hedströen file technique with the reciprocal reverse cutting technique of the Wave One® Gold Primary file and the use of E.D.T.A. 24% gel allowed the fractured file fragment to be overcome in the middle third of the mesiobuccal canal of tooth 17, promoting hermetic sealing, cleaning, modeling and satisfactory filling. (AU)

Fast NMR method to probe solvent accessibility and disordered regions in proteins.

Understanding protein structure and dynamics, which govern key cellular processes, is crucial for basic and applied research. Intrinsically disordered protein (IDP) regions display multifunctionality via alternative transient conformations, being key players in disease mechanisms. IDP regions are abundant, namely in small viruses, allowing a large number of functions out of a small proteome. The relation between protein function and structure is thus now seen from a different perspective: as IDP regions enable transient structural arrangements, each conformer can play different roles within the cell. However, as IDP regions are hard and time-consuming to study via classical techniques (optimized for globular proteins with unique conformations), new methods are required. Here, employing the dengue virus (DENV) capsid (C) protein and the immunoglobulin-binding domain of streptococcal protein G, we describe a straightforward NMR method to differentiate the solvent accessibility of single amino acid N-H groups in structured and IDP regions. We also gain insights into DENV C flexible fold region biological activity. The method, based on minimal pH changes, uses the well-established 1H-15N HSQC pulse sequence and is easily implementable in current protein NMR routines. The data generated are simple to interpret, with this rapid approach being an useful first-choice IDPs characterization method.

First insights of peptidoglycan amidation in Gram-positive bacteria - the high-resolution crystal structure of Staphylococcus aureus glutamine amidotransferase GatD.

Gram-positive bacteria homeostasis and antibiotic resistance mechanisms are dependent on the intricate architecture of the cell wall, where amidated peptidoglycan plays an important role. The amidation reaction is carried out by the bi-enzymatic complex MurT-GatD, for which biochemical and structural information is very scarce. In this work, we report the first crystal structure of the glutamine amidotransferase member of this complex, GatD from Staphylococcus aureus, at 1.85 Å resolution. A glutamine molecule is found close to the active site funnel, hydrogen-bonded to the conserved R128. In vitro functional studies using 1H-NMR spectroscopy showed that S. aureus MurT-GatD complex has glutaminase activity even in the absence of lipid II, the MurT substrate. In addition, we produced R128A, C94A and H189A mutants, which were totally inactive for glutamine deamidation, revealing their essential role in substrate sequestration and catalytic reaction. GatD from S. aureus and other pathogenic bacteria share high identity to enzymes involved in cobalamin biosynthesis, which can be grouped in a new sub-family of glutamine amidotransferases. Given the ubiquitous presence of GatD, these results provide significant insights into the molecular basis of the so far undisclosed amidation mechanism, contributing to the development of alternative therapeutics to fight infections.

Epitope mapping of imidazolium cations in ionic liquid-protein interactions unveils the balance between hydrophobicity and electrostatics towards protein destabilisation.

We investigated imidazolium-based ionic liquid (IL) interactions with human serum albumin (HSA) to discern the level of cation interactions towards protein stability. STD-NMR spectroscopy was used to observe the imidazolium IL protons involved in direct binding and to identify the interactions responsible for changes in Tm as accessed by differential scanning calorimetry (DSC). Cations influence protein stability less than anions but still significantly. It was found that longer alkyl side chains of imidazolium-based ILs (more hydrophobic) are associated with a higher destabilisation effect on HSA than short-alkyl groups (less hydrophobic). The reason for such destabilisation lies on the increased surface contact area of the cation with the protein, particularly on the hydrophobic contacts promoted by the terminus of the alkyl chain. The relevance of the hydrophobic contacts is clearly demonstrated by the introduction of a polar moiety in the alkyl chain: a methoxy or alcohol group. Such structural modification reduces the degree of hydrophobic contacts with HSA explaining the lesser extent of protein destabilisation when compared to longer alkyl side chain groups: above [C2mim](+). Competition STD-NMR experiments using [C2mim](+), [C4mim](+) and [C2OHmim](+) also validate the importance of the hydrophobic interactions. The combined effect of cation and anion interactions was explored using (35)Cl NMR. Such experiments show that the nature of the cation has no influence on the anion-protein contacts, still the nature of the anion modulates the cation-protein interaction. Herein we propose that more destabilising anions are likely to be a result of a partial contribution from the cation as a direct consequence of the different levels of interaction (cation-anion pair and cation-protein).