Breaking ties: The antitumour protein, neocarzinostatin (NCS), is one of the few drug-carrying proteins used in human therapeutics. However, the presence of disulfide bonds limits this protein's potential development for many applications. This study describes a generic directed-evolution approach starting from NCS-3.24 (shown in the figure complexed with two testosterone molecules) to engineer stable disulfide-free NCS variants suitable for a variety of purposes, including intracellular applications.The chromoprotein neocarzinostatin (NCS) has been intensively studied for its antitumour properties. It has recently been redesigned as a potential drug-carrying scaffold. A potential limit of this protein scaffold, especially for intracellular applications, is the presence of disulfide bonds. The objective of this work was to create a disulfide-free NCS-derived scaffold. A generic targeted approach was developed by using directed evolution methods. As a starting point we used a previously engineered NCS variant in which a hapten binding site had been created. A library was then generated in which cysteine Cys88 and Cys93 and neighbouring residues were randomly substituted. Variants that preserved the hapten binding function were selected by phage display and further screened by colony filtration methods. Several sequences with common features emerged from this process. The corresponding proteins were expressed, purified and their biophysical properties characterised. How these selected sequences rescued folding ability and stability of the disulfide-free protein was carefully examined by using calorimetry and the results were interpreted with molecular simulation techniques.
Introduction. Group A Rotavirus (RVA) is known to be a major cause of acute gastroenteritis (AGE) in children but its role as a potential pathogen in immunocompetent adults is probably underestimated.Aim. To compare RVA infections in patients from different age groups.Methodology. Fecal samples were collected from patients aged from birth to 65 years, hospitalized or consulting for AGE between 2015 and 2017. All samples were screened by RT-PCR for the detection of VP6 gene specific of RVA. RVA-positive samples were VP7 and VP4 genotyped using multiplex semi-nested RT-PCR. Full-length VP7 gene of G9-positive strains were sequenced and submitted for phylogenetic analysis.Results. Of 1371 stool specimens collected from children (<5 years; n=454), older children (5 to <15 years; n=316) and adults (15-65 years; n=601), 165 (12.0 %) were RVA-positive. RVA detection rates were significantly higher in children and adults than in older children (15.8 % and 12.1 Vs 6.3 %, respectively; P<0.001). While RVA infections were mostly detected during the coldest months in children, they were observed all year-round in patients aged >5 years. Although G1P[8] remained the most prevalent combination (41.7 %) detected in children, G9P[8] strains widely predominated in adults (58.1 %), followed by G2P[4] (12.9 %). All characterized G9 strains clustered in the modern lineage III.Conclusion. RVA play an important role in AGE not only in children but also in adults. The findings of a wide G9 predominance in patients >5 years highlights the need for continuing surveillance in both pediatric and mature populations.
Introduction. Respiratory syncytial virus (RSV) is the most frequently identified viral agent in children with lower respiratory tract infection (LRTI). No data are available to date regarding RSV genotypes circulating in Tunisia. Aim. The aim of the present study was to investigate the genetic variability of the glycoprotein G gene in Tunisian RSV strains. Methodology. Nasopharyngeal aspirates were collected from infants hospitalized for LRTI in five Tunisian hospitals. All specimens were screened for RSV by a direct immunofluorescence assay (DIFA). To molecularly characterize Tunisian RSV strains, a phylogenetic analysis was conducted. Randomly selected positive samples were subjected to reverse transcription PCR amplifying the second hyper-variable region (HVR2) of the G gene. Results. Among a total of 1417 samples collected between 2015 and 2018, 394 (27.8 %) were positive for RSV by DIFA. Analysis of 61 randomly selected RSV strains revealed that group A RSV (78.7 %) predominated during the period of study as compared to group B RSV (21.3 %). The phylogenetic analysis showed that two genotypes of RSV-A were co-circulating: the ON1 genotype with a 72-nt duplication in HVR2 of the G gene was predominant (98.0 % of RSV-A strains), while one RSV-A strain clustered with the NA1 genotype (2.0 %). Concerning Tunisian group B RSV strains, all sequences contained a 60-nt insertion in HVR2 and a clustered BA10 genotype. Conclusion. These data suggest that RSV-A genotype ON1 and RSV-B genotype BA10, both with duplications in the G gene, were widely circulating in the Central coastal region of Tunisia between 2015 and 2018.
The aim of the present study was to report the molecular characterization of human group A rotaviruses (RVAs) circulating in Tunisia. Stool specimens were collected from children under 5 years of age who had been hospitalized or were consulting for gastroenteritis in Tunisian hospitals between 2015 and 2017. All samples were screened by reverse-transcription polymerase chain reaction (RT-PCR) for the detection of the VP6 gene specific for RVA. RVA-positive samples were further analysed for G/P genotyping by semi-nested multiplex RT-PCR. Among 454 tested samples, 72 (15.8 %) were positive for RVA. G1P[8] was the most prevalent detected strain (41.7%), followed by G9P[8] (32.8%), G2P[4] (7.5%), G12P[8] (7.5%), G1P[6] (3.0%), G2P[8] (1.5%) and G3P[8] (1.5%), with mixed infections in 4.5 % of cases. In the absence of a national anti-rotavirus vaccination strategy, RVAs remain the primary aetiological agent for gastroenteritis in Tunisian children.
Protein flexibility is essential for enzymatic function, ligand binding, and protein–protein or protein–nucleic acid interactions. Normal mode analysis has increasingly been shown to be well suited for studying such flexibility, as it can be used to identify favorable structural deformations that correspond to functional motions. However, normal modes are strictly relevant to a single structure, reflecting a particular minimum on a complex energy surface, and are thus susceptible to artifacts. We describe a new theoretical framework for determining "consensus" normal modes from a set of related structures, such as those issuing from a short molecular dynamics simulation. This approach is more robust than standard normal mode analysis, and provides higher collectivity and symmetry properties. In an application to HIV-1 protease, the low-frequency consensus modes describe biologically relevant motions including flap opening and closing that can be used in interpreting structural changes accompanying the binding of widely differing inhibitors.
Small G-proteins of the superfamily Ras function as molecular switches, interacting with different cellular partners according to their activation state. G-protein activation involves the dissociation of bound GDP and its replacement by GTP, in an exchange reaction that is accelerated and regulated in the cell by guanine-nucleotide exchange factors (GEFs). Large conformational changes accompany the exchange reaction, and our understanding of the mechanism is correspondingly incomplete. However, much knowledge has been derived from structural studies of blocked or inactive mutant GEFs, which presumably closely represent intermediates in the exchange reaction and yet which are by design incompetent for carrying out the nucleotide exchange reaction. In this study we have used comparative modelling to recreate an exchange-competent form of a late, pre-GDP-ejection intermediate species in Arf1, a well-characterized small G-protein. We extensively characterized three distinct models of this intermediate using molecular dynamics simulations, allowing us to address ambiguities related to the mutant structural studies. We observed in particular the unfavorable nature of Mg2+ associated forms of the complex and the establishment of closer Arf1-GEF contacts in its absence. The results of this study shed light on GEF-mediated activation of this small G protein and on predicting the fate of the Mg ion at a critical point in the exchange reaction. The structural models themselves furnish additional targets for interfacial inhibitor design, a promising direction for exploring potentially druggable targets with high biological specificity.
