The osmolyte trimethylamine‐N‐oxide stabilizes the Fyn SH3 domain without altering the structure of its folding transition state

Trimethylamine‐N‐oxide (TMAO) is a naturally occurring osmolyte that stabilizes proteins against denaturation. Although the impact of TMAO on the folding thermodynamics of many proteins has been well characterized, far fewer studies have investigated its effects on protein folding kinetics. In particular, no previous studies have used Φ‐value analysis to determine whether TMAO may alter the structure of the folding transition state. Here we have measured the effects on folding kinetics of 16 different amino acid substitutions distributed across the structure of the Fyn SH3 domain both in the presence and absence of TMAO. The folding and unfolding rates in TMAO, on average, improved to equivalent degrees, with a twofold increase in the protein folding rate accompanied by a twofold decrease in the unfolding rate. Importantly, TMAO caused little alteration to the Φ‐values of the mutants tested, implying that this compound minimally perturbs the folding transition state structure. Furthermore, the solvent accessibility of the transition state was not altered as reflected in an absence of a TMAO‐induced change in the denaturant β factors. Through TMAO‐induced folding studies, a β factor of 0.5 was calculated for this compound, suggesting that the protein backbone, which is the target of action of TMAO, is 50% exposed in the transition state as compared to the native state. This finding is consistent with the equivalent effects of TMAO on the folding and unfolding rates. Through thermodynamic analysis of mutants, we also discovered that the stabilizing effect of TMAO is lessened with increasing temperature.     

A de novo redesign of the WW domain

We have used a sequence prediction algorithm and a novel sampling method to design protein sequences for the WW domain, a small beta-sheet motif. The procedure, referred to as SPANS, designs sequences to be compatible with an ensemble of closely related polypeptide backbones, mimicking the inherent flexibility of proteins. Two designed sequences (termed SPANS-WW1 and SPANS-WW2), using only naturally occurring L-amino acids, were selected for study and the corresponding polypeptides were prepared in Escherichia coli. Circular dichroism data suggested that both purified polypeptides adopted secondary structure features related to those of the target without the aid of disulfide bridges or bound cofactors. The structure exhibited by SPANS-WW2 melted cooperatively by raising the temperature of the solution. Further analysis of this polypeptide by proton nuclear magnetic resonance spectroscopy demonstrated that at 5 degrees C, it folds into a structure closely resembling a natural WW domain. This achievement constitutes one of a small number of successful de novo protein designs through fully automated computational methods and highlights the feasibility of including backbone flexibility in the design strategy.     

Recognition of RNA cap in the Wesselsbron virus NS5 methyltransferase domain: implications for RNA-capping mechanisms in Flavivirus

The mRNA-capping process starts with the conversion of a 5′-triphosphate end into a 5′-diphosphate by an RNA triphosphatase, followed by the addition of a guanosine monophosphate unit in a 5′-5′ phosphodiester bond by a guanylyltransferase. Methyltransferases are involved in the third step of the process, transferring a methyl group from S-adenosyl-l-methionine to N7-guanine (cap 0) and to the ribose 2′OH group (cap 1) of the first RNA nucleotide; capping is essential for mRNA stability and proper replication. In the genus Flavivirus, N7-methyltransferase and 2′O-methyltransferase activities have been recently associated with the N-terminal domain of the viral NS5 protein. In order to further characterize the series of enzymatic reactions that support capping, we analyzed the crystal structures of Wesselsbron virus methyltransferase in complex with the S-adenosyl-l-methionine cofactor, S-adenosyl-l-homocysteine (the product of the methylation reaction), Sinefungin (a molecular analogue of the enzyme cofactor), and three different cap analogues (GpppG, ^N7MeGpppG, and ^N7MeGpppA). The structural results, together with those on other flaviviral methyltransferases, show that the capped RNA analogues all bind to an RNA high-affinity binding site. However, lack of specific interactions between the enzyme and the first nucleotide of the RNA chain suggests the requirement of a minimal number of nucleotides following the cap to strengthen protein/RNA interaction. Our data also show that, following incubation with guanosine triphosphate, Wesselsbron virus methyltransferase displays a guanosine monophosphate molecule covalently bound to residue Lys28, hinting at possible implications for the transfer of a guanine group to ppRNA. The structures of the Wesselsbron virus methyltransferase complexes obtained are discussed in the context of a model for N7-methyltransferase and 2′O-methyltransferase activities.     

The Trimeric Major Capsid Protein of Mavirus is stabilized by its Interlocked N-termini Enabling Core Flexibility for Capsid Assembly

Icosahedral viral capsids assemble with high fidelity from a large number of identical buildings blocks. The mechanisms that enable individual capsid proteins to form stable oligomeric units (capsomers) while affording structural adaptability required for further assembly into capsids are mostly unknown.Understanding these mechanisms requires knowledge of the capsomers’ dynamics, especially for viruses where no additional helper proteins are needed during capsid assembly like for the Mavirus virophage that despite its complexity (triangulation number T = 27) can assemble from its major capsid protein (MCP) alone. This protein forms the basic building block of the capsid namely a trimer (MCP₃) of double-jelly roll protomers with highly intertwined N-terminal arms of each protomer wrapping around the other two at the base of the capsomer, secured by a clasp that is formed by part of the C-terminus.Probing the dynamics of the capsomer with HDX mass spectrometry we observed differences in conformational flexibility between functional elements of the MCP trimer. While the N-terminal arm and clasp regions show above average deuterium incorporation, the two jelly-roll units in each protomer also differ in their structural plasticity, which might be needed for efficient assembly. Assessing the role of the N-terminal arm in maintaining capsomer stability showed that its detachment is required for capsomer dissociation, constituting a barrier towards capsomer monomerisation. Surprisingly, capsomer dissociation was irreversible since it was followed by a global structural rearrangement of the protomers as indicated by computational studies showing a rearrangement of the N-terminus blocking part of the capsomer forming interface.     

Deciphering Spatial Protein–Protein Interactions in Brain Using Proximity Labeling

Cellular biomolecular complexes including protein–protein, protein–RNA, and protein–DNA interactions regulate and execute most biological functions. In particular in brain, protein–protein interactions (PPIs) mediate or regulate virtually all nerve cell functions, such as neurotransmission, cell–cell communication, neurogenesis, synaptogenesis, and synaptic plasticity. Perturbations of PPIs in specific subsets of neurons and glia are thought to underly a majority of neurobiological disorders. Therefore, understanding biological functions at a cellular level requires a reasonably complete catalog of all physical interactions between proteins. An enzyme-catalyzed method to biotinylate proximal interacting proteins within 10 to 300 nm of each other is being increasingly used to characterize the spatiotemporal features of complex PPIs in brain. Thus, proximity labeling has emerged recently as a powerful tool to identify proteomes in distinct cell types in brain as well as proteomes and PPIs in structures difficult to isolate, such as the synaptic cleft, axonal projections, or astrocyte–neuron junctions. In this review, we summarize recent advances in proximity labeling methods and their application to neurobiology.     

Gene expression signature of human HepG2 cell line

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and is associated with various clinico-pathological characteristics such as genetic mutations and viral infections. Therefore, numerous laboratories look out for identifying always new putative markers for the improvement of HCC diagnosis/prognosis. Many molecular profiling studies investigated gene expression changes related to HCC. HepG2 represents a pure cell line of human liver carcinoma, often used as HCC model due to the absence of viral infection. In this study we compare gene expression profiles associated with HepG2 (as HCC model) and normal hepatocyte cells by microarray technology. Hierarchical cluster analysis of genes evidenced that 2646 genes significantly down-regulated in HepG2 cells compared to hepatocytes whereas a further 3586 genes significantly up-regulated. By using the Ingenuity Pathway Analysis (IPA) program, we have classified the genes that were differently expressed and studied the functional networks correlating these genes in the complete human interactome. Moreover, to confirm the differentially expressed genes as well as the reliability of our microarray data, we performed a quantitative Real time RT-PCR analysis on 9 up-regulated and 11 down-regulated genes, respectively. In conclusion this work i) provides a gene signature of human hepatoma cells showing genes that change their expression as a consequence of liver cancer in the absence of any genetic mutations or viral infection, ii) evidences new differently expressed genes found in our signature compared to previous published studies and iii) suggests some genes on which to focus future studies to understand if they can be used to improve the HCC prognosis/diagnosis.     

Array CGH analysis of a cohort of Russian patients with intellectual disability

The use of array comparative genomic hybridization (array CGH) as a diagnostic tool in molecular genetics has facilitated the identification of many new microdeletion/microduplication syndromes (MMSs). Furthermore, this method has allowed for the identification of copy number variations (CNVs) whose pathogenic role has yet to be uncovered. Here, we report on our application of array CGH for the identification of pathogenic CNVs in 79 Russian children with intellectual disability (ID). Twenty-six pathogenic or likely pathogenic changes in copy number were detected in 22 patients (28%): 8 CNVs corresponded to known MMSs, and 17 were not associated with previously described syndromes. In this report, we describe our findings and comment on genes potentially associated with ID that are located within the CNV regions.     

Design,synthesis and molecular docking of thiazolidinedione based benzene sulphonamide derivatives containing pyrazole core as potential anti-diabetic agents

We herein report the design, synthesis and molecular docking studies of 2,4-thiazolidinedione derivatives containing benzene sulphonyl group which are docked against the Peroxisome Proliferator Activated Receptor (PPARγ) target. Compound 7p was most effective in lowering the blood glucose level as compared to standard drugs pioglitazone and rosiglitazone. Compound 7p exhibited potent PPAR-γ transactivation of 61.2% with 1.9 folds increase in gene expression. In molecular docking studies 7p showed excellent interactions with amino acids TYR 473, SER 289, HIE 449, TYR 327, ARG 288, MET 329 and LEU 228. Compound 7p did not cause any damage to the liver without any noteworthy weight gain and may be considered as promising candidates for the development of new antidiabetic agents.