A comparison of four different conformations adopted by human telomeric G‐quadruplex using computer simulations

The telomeric G‐quadruplexes for their unique structural features are considered as potential anticancer drug targets. These, however, exhibit structural polymorphism as different topology types for the intra‐molecular G‐quadruplexes from human telomeric G‐rich sequences have been reported based on NMR spectroscopy and X‐ray crystallography. These techniques provide detailed atomic‐level information about the molecule but relative conformational stability of the different topologies remains unsolved. Therefore, to understand the conformational preference, we have carried out quantum chemical calculations on G‐quartets; used all‐atom molecular dynamics (MD) simulations and steered molecular dynamics (SMD) simulations to characterize the four human telomeric G‐quadruplex topologies based on its G‐tetrad core‐types, viz., parallel, anti‐parallel, mixed‐(3 + 1)‐form1 and mixed‐(3 + 1)‐form2. We have also studied a non‐telomeric sequence along with these telomeric forms giving a comparison between the two G‐rich forms. The structural properties such as base pairing, stacking geometry and backbone conformations have been analyzed. The quantum calculations indicate that presence of a sodium ion inside the G‐tetrad plane or two potassium ions on both sides of the plane give it an overall planarity which is much needed for good stacking to form a helix. MD simulations indicate that capping of the G‐tetrad core by the TTA loops keep the terminal guanine bases away from water. The SMD simulations along with equilibrium MD studies indicate that the parallel and non‐telomeric forms are comparatively less stable. We could come to the conclusion that the anti‐parallel form and also the mixed‐(3 + 1)‐form1 topology are most likely to represent the major conformation., 2016. © 2015 Wiley Periodicals, Inc. Biopolymers 105: 83–99, 2016     

An assay for 26S proteasome activity based on fluorescence anisotropy measurements of dye-labeled protein substrates

The 26S proteasome is the molecular machine at the center of the ubiquitin proteasome system and is responsible for adjusting the concentrations of many cellular proteins. It is a drug target in several human diseases, and assays for the characterization of modulators of its activity are valuable. The 26S proteasome consists of two components: a core particle, which contains the proteolytic sites, and regulatory caps, which contain substrate receptors and substrate processing enzymes, including six ATPases. Current high-throughput assays of proteasome activity use synthetic fluorogenic peptide substrates that report directly on the proteolytic activity of the proteasome, but not on the activities of the proteasome caps that are responsible for protein recognition and unfolding. Here, we describe a simple and robust assay for the activity of the entire 26S proteasome using fluorescence anisotropy to follow the degradation of fluorescently labeled protein substrates. We describe two implementations of the assay in a high-throughput format and show that it meets the expected requirement of ATP hydrolysis and the presence of a canonical degradation signal or degron in the target protein.     

Reversible HuR‐microRNA binding controls extracellular export of miR‐122 and augments stress response

microRNAs (miRNAs), the tiny but stable regulatory RNAs in metazoan cells, can undergo selective turnover in presence of specific internal and external cues to control cellular response against the changing environment. We have observed reduction in cellular miR‐122 content, due to their accelerated extracellular export in human hepatic cells starved for small metabolites including amino acids. In this context, a new role of human ELAV protein HuR has been identified. HuR, a negative regulator of miRNA function, accelerates extracellular vesicle (EV)‐mediated export of miRNAs in human cells. In stressed cells, HuR replaces miRNPs from target messages and is both necessary and sufficient for the extracellular export of corresponding miRNAs. HuR could reversibly bind miRNAs to replace them from Ago2 and subsequently itself gets freed from bound miRNAs upon ubiquitination. The ubiquitinated form of HuR is predominantly associated with multivesicular bodies (MVB) where HuR‐unbound miRNAs also reside. These MVB‐associated pool of miRNAs get exported out via EVs thereby delimiting cellular miR‐122 level during starvation. Therefore, by modulating extracellular export of miR‐122, HuR could control stress response in starved human hepatic cells.     

Prdm9 and Meiotic Cohesin Proteins Cooperatively Promote DNA Double-Strand Break Formation in Mammalian Spermatocytes

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The cyclic AMP cascade is altered in the fragile X nervous system

Fragile X syndrome (FX), the most common heritable cause of mental retardation and autism, is a developmental disorder characterized by physical, cognitive, and behavioral deficits. FX results from a trinucleotide expansion mutation in the fmr1 gene that reduces levels of fragile X mental retardation protein (FMRP). Although research efforts have focused on FMRP's impact on mGluR signaling, how the loss of FMRP leads to the individual symptoms of FX is not known. Previous studies on human FX blood cells revealed alterations in the cyclic adenosine 3', 5'-monophosphate (cAMP) cascade. We tested the hypothesis that cAMP signaling is altered in the FX nervous system using three different model systems. Induced levels of cAMP in platelets and in brains of fmr1 knockout mice are substantially reduced. Cyclic AMP induction is also significantly reduced in human FX neural cells. Furthermore, cAMP production is decreased in the heads of FX Drosophila and this defect can be rescued by reintroduction of the dfmr gene. Our results indicate that a robust defect in cAMP production in FX is conserved across species and suggest that cAMP metabolism may serve as a useful biomarker in the human disease population. Reduced cAMP induction has implications for the underlying causes of FX and autism spectrum disorders. Pharmacological agents known to modulate the cAMP cascade may be therapeutic in FX patients and can be tested in these models, thus supplementing current efforts centered on mGluR signaling.     

Deciphering binding affinity,energetics, and base specificity of plant alkaloid Harmane with AT and GC hairpin duplex DNA

Insights into binding efficacy and thermodynamic aspects of small molecules are important for rational drug designing and development. Here, the interaction of Harmane (Har), a very important bioactive indole alkaloid, with AT and GC hairpin duplex−DNAs has been reported using various biophysical tools. Detailed molecular mechanism with special emphasis on binding nature, base specificity, and thermodynamics have been elucidated via probing nucleic acids with varying base compositions. Har bound to both the DNA strands exhibited hypochromic effect in absorbance whereas bathochromic and hypochromic effects in fluorescence spectra. The binding constants estimated were in the order of 10⁵ M⁻¹ (higher for GC sequence compared with AT) with 1:1 stoichiometry. Noncooperative binding mode has been observed via intercalation in both the cases. The thermodynamic profile was obtained from temperature-dependent fluorescence experiments. Both Har–AT and Har–GC complexations were exothermic in nature associated with positive entropy and negative enthalpy changes. Salt-dependent studies revealed that the binding interaction was governed by nonpolyelectrolytic and hydrophobic interaction forces. The ligand-induced structural perturbation of the DNA structures was evident from the circular dichroism data. Molecular modelling data indicated towards the involvement of hydrophobic forces and hydrogen bonding.     

The aggregation state of rhodanese during folding influences the ability of GroEL to assist reactivation

The in vitro folding of rhodanese involves a competition between formation of properly folded enzyme and off-pathway inactive species. Co-solvents like glycerol or low temperature, e.g. refolding at 10 degrees C, successfully retard the off-pathway formation of large inactive aggregates, but the process does not yield 100% active enzyme. These data suggest that mis-folded species are formed from early folding intermediates. GroEL can capture early folding intermediates, and it loses the ability to capture and reactivate rhodanese if the enzyme is allowed first to spontaneously fold for longer times before it is presented to GroEL, a process that leads to the formation of unproductive intermediates. In addition, GroEL cannot reverse large aggregates once they are formed, but it could capture some folding intermediates and activate them, even though they are not capable of forming active enzyme if left to spontaneous refolding. The interaction between GroEL and rhodanese substantially but not completely inhibits intra-protein inactivation, which is responsible for incomplete activation during unassisted refolding. Thus, GroEL not only decreases aggregation, but it gives the highest reactivation of any method of assistance. The results are interpreted using a previously suggested model based on studies of the spontaneous folding of rhodanese (Gorovits, B. M., McGee, W. A., and Horowitz, P. M. (1998) Biochim. Biophys. Acta 1382, 120--128 and Panda, M., Gorovits, B. M., and Horowitz, P. M. (2000) J. Biol. Chem. 275, 63--70).