Equilibrium model selection: dTTP induced R1 dimerization

<p>Abstract</p> <p>Background</p> <p>Biochemical equilibria are usually modeled iteratively: given one or a few fitted models, if there is a lack of fit or over fitting, a new model with additional or fewer parameters is then fitted, and the process is repeated. The problem with this approach is that different analysts can propose and select different models and thus extract different binding parameter estimates from the same data. An alternative is to first generate a comprehensive standardized list of plausible models, and to then fit them exhaustively, or semi-exhaustively.</p> <p>Results</p> <p>A framework is presented in which equilibriums are modeled as pairs (<it>g</it>, <it>h</it>) where <it>g </it>= 0 maps total reactant concentrations (system inputs) into free reactant concentrations (system states) which <it>h </it>then maps into expected values of measurements (system outputs). By letting dissociation constants <it>K</it>_{<it>d </it>}be either freely estimated, infinity, zero, or equal to other <it>K</it>_<it>d</it>, and by letting undamaged protein fractions be either freely estimated or 1, many <it>g </it>models are formed. A standard space of <it>g </it>models for ligand-induced protein dimerization equilibria is given. Coupled to an <it>h </it>model, the resulting (<it>g</it>, <it>h</it>) were fitted to dTTP induced R1 dimerization data (R1 is the large subunit of ribonucleotide reductase). Models with the fewest parameters were fitted first. Thereafter, upon fitting a batch, the next batch of models (with one more parameter) was fitted only if the current batch yielded a model that was better (based on the Akaike Information Criterion) than the best model in the previous batch (with one less parameter). Within batches models were fitted in parallel. This semi-exhaustive approach yielded the same best models as an exhaustive model space fit, but in approximately one-fifth the time.</p> <p>Conclusion</p> <p>Comprehensive model space based biochemical equilibrium model selection methods are realizable. Their significance to systems biology as mappings of data into mathematical models warrants their development.</p

Competitive and Cooperative Interactions Mediate RNA Transfer from Herpesvirus Saimiri ORF57 to the Mammalian Export Adaptor ALYREF

The essential herpesvirus adaptor protein HVS ORF57, which has homologs in all other herpesviruses, promotes viral mRNA export by utilizing the cellular mRNA export machinery. ORF57 protein specifically recognizes viral mRNA transcripts, and binds to proteins of the cellular transcription-export (TREX) complex, in particular ALYREF. This interaction introduces viral mRNA to the NXF1 pathway, subsequently directing it to the nuclear pore for export to the cytoplasm. Here we have used a range of techniques to reveal the sites for direct contact between RNA and ORF57 in the absence and presence of ALYREF. A binding site within ORF57 was characterized which recognizes specific viral mRNA motifs. When ALYREF is present, part of this ORF57 RNA binding site, composed of an a-helix, binds preferentially to ALYREF. This competitively displaces viral RNA from the a-helix, but contact with RNA is still maintained by a flanking region. At the same time, the flexible N-terminal domain of ALYREF comes into contact with the viral RNA, which becomes engaged in an extensive network of synergistic interactions with both ALYREF and ORF57. Transfer of RNA to ALYREF in the ternary complex, and involvement of individual ORF57 residues in RNA recognition, were confirmed by UV cross-linking and mutagenesis. The atomic-resolution structure of the ORF57-ALYREF interface was determined, which noticeably differed from the homologous ICP27-ALYREF structure. Together, the data provides the first site-specific description of how viral mRNA is locked by a herpes viral adaptor protein in complex with cellular ALYREF, giving herpesvirus access to the cellular mRNA export machinery. The NMR strategy used may be more generally applicable to the study of fuzzy protein-protein-RNA complexes which involve flexible polypeptide regions

A morphological evaluation of botulinum neurotoxin A injections into the detrusor muscle using magnetic resonance imaging

OBJECTIVES: Although botulinum neurotoxin type A (BoNT/A) intradetrusor injections are a recommended therapy for neurogenic detrusor overactivity (NDO), refractory to antimuscarinic drugs, a standardisation of injection technique is missing. Furthermore, some basic questions are still unanswered, as where the toxin solution exactly spreads after injection. Therefore, we investigated the distribution of the toxin solution after injection into the bladder wall, using magnet resonance imaging (MRI). METHODS: Six patients with NDO were recruited. Three of six patients received 300 U of BoNT/A + contrast agent distributed over 30 injection sites (group 1). The other three patients received 300 U of BoNT/A + contrast agent distributed over 10 injection sites (group 2). Immediately after injection, MRI of the pelvis was performed. The volume of the detrusor and the total volume of contrast medium inside and outside the bladder wall were calculated. RESULTS: In all patients, a small volume (mean 17.6%) was found at the lateral aspects of the bladder dome in the extraperitoneal fat tissue, whereas 82.4% of the injected volume reached the target area (detrusor). In both groups there was a similar distribution of the contrast medium in the target area. A mean of 33.3 and 25.3% of the total detrusor volume was covered in group 1 and 2, respectively. Six weeks after injection, five of six patients were continent and showed no detrusor overactivity in the urodynamic follow-up. No systemic side effects were observed. CONCLUSIONS: Our results provide morphological arguments that the currently used injection techniques are appropriate and safe

Nucleic acid recognition and antiviral activity of 1,4-substituted terphenyl compounds mimicking all faces of the HIV-1 Rev protein positively-charged α-helix

Small synthetic molecules mimicking the three-dimensional structure of α-helices may find applications as inhibitors of therapeutically relevant protein-protein and protein-nucleic acid interactions. However, the design and use of multi-facial helix mimetics remains in its infancy. Here we describe the synthesis and application of novel bilaterally substituted p-terphenyl compounds containing positively-charged aminoalkyl groups in relative 1,4 positions across the aromatic scaffold. These compounds were specifically designed to mimic all faces of the arginine-rich α-helix of the HIV-1 protein Rev, which forms deeply embedded RNA complexes and plays key roles in the virus replication cycle. Two of these molecules recognized the Rev site in the viral RNA and inhibited the formation of the RRE-Rev ribonucleoprotein complex, a currently unexploited target in HIV chemotherapy. Cellular assays revealed that the most active compounds blocked HIV-1 replication with little toxicity, and likely exerted this effect through a multi-target mechanism involving inhibition of viral LTR promoter-dependent transcription and Rev function. Further development of this scaffold may open new avenues for targeting nucleic acids and may complement current HIV therapies, none of which involve inhibitors interfering with the gene regulation processes of the virus.This project was supported by Ministerio de Economía y Competitividad of Spain (Grants BFU2012–30770 and BFU2015–65103-R to J.G.; CTQ2013-43310 and CTQ2017-84249-P to S.F. and FIS PI16CIII/0034 to J.A.; and FPU15/01485 predoctoral fellowship to D.M.S.), Generalitat Valenciana of Spain (FPA/2015/014 and APOTIP/2016/A007 to J.G. and PROMETEOII/2014/073 to S.F.), the Spanish AIDS Research Network (RD16CIII/0002/0001-ISCIII–FEDER to J.A.), Universidad Católica de Valencia (2017-114-001 and 2018-114-001 to J.G.), and European AIDS Vaccine Initiative 2020 (ID 681137 to J.A.). The authors thank Ainhoa Sánchez for carrying out initial fluorescence anisotropy experiments, Ángel Cantero-Camacho for designing and testing the primers used to amplify LTRc, and Jerónimo Bravo and Antonio Pineda for facilitating access to ITC equipment. Plasmid pLTR(HTLV)-luc (pGL4.20-U3R) was kindly donated by Thomas Kress.S