Unraveling the impact of the p.R107L mutation on the structure and function of human αB-Crystallin: Implications for cataract formation.

To date, several pathogenic mutations have been identified in the primary structure of human α-Crystallin, frequently involving the substitution of arginine with a different amino acid. These mutations can lead to the incidence of cataracts and myopathy. Recently, an important cataract-associated mutation has been reported in the functional α-Crystallin domain (ACD) of human αB-Crystallin protein, where arginine 107 (R107) is replaced by a leucine. In this study, we investigated the structure, chaperone function, stability, oligomerization, and amyloidogenic properties of the p.R107L human αB-Crystallin using a number of different techniques. Our results suggest that the p.R107L mutation can cause significant changes in the secondary, tertiary, and quaternary structures of αB-Crystallin. This cataractogenic mutation led to the formation of protein oligomers with larger sizes than the wild-type protein and reduced the chemical and thermal stability of the mutant chaperone. Both fluorescence and microscopic assessments indicated that this mutation significantly altered the amyloidogenic properties of human αB-Crystallin. Furthermore, the mutant protein indicated an attenuated in vitro chaperone activity. The molecular dynamics (MD) simulation confirmed the experimental results and indicated that p.R107L mutation could alter the proper conformation of human αB-Crystallin dimers. In summary, our results indicated that the p.R107L mutation could promote the formation of larger oligomers, diminish the stability and chaperone activity of human αB-Crystallin, and these changes, in turn, can play a crucial role in the development of cataract disorder.

Hybrid DNA/RNA nanostructures with 2'-5' linkages.

Nucleic acid nanostructures with different chemical compositions have shown utility in biological applications as they provide additional assembly parameters and enhanced stability. The naturally occurring 2'-5' linkage in RNA is thought to be a prebiotic analogue and has potential use in antisense therapeutics. Here, we report the first instance of DNA/RNA motifs containing 2'-5' linkages. We synthesized and incorporated RNA strands with 2'-5' linkages into different DNA motifs with varying number of branch points (a duplex, four arm junction, double crossover motif and tensegrity triangle motif). Using experimental characterization and molecular dynamics simulations, we show that hybrid DNA/RNA nanostructures can accommodate interspersed 2'-5' linkages with relatively minor effect on the formation of these structures. Further, the modified nanostructures showed improved resistance to ribonuclease cleavage, indicating their potential use in the construction of robust drug delivery vehicles with prolonged stability in physiological conditions.

Base-pair conformational switch modulates miR-34a targeting of Sirt1 mRNA.

MicroRNAs (miRNAs) regulate the levels of translation of messenger RNAs (mRNAs). At present, the major parameter that can explain the selection of the target mRNA and the efficiency of translation repression is the base pairing between the 'seed' region of the miRNA and its counterpart mRNA1. Here we use R1ρ relaxation-dispersion nuclear magnetic resonance2 and molecular simulations3 to reveal a dynamic switch-based on the rearrangement of a single base pair in the miRNA-mRNA duplex-that elongates a weak five-base-pair seed to a complete seven-base-pair seed. This switch also causes coaxial stacking of the seed and supplementary helix fitting into human Argonaute 2 protein (Ago2), reminiscent of an active state in prokaryotic Ago4,5. Stabilizing this transient state leads to enhanced repression of the target mRNA in cells, revealing the importance of this miRNA-mRNA structure. Our observations tie together previous findings regarding the stepwise miRNA targeting process from an initial 'screening' state to an 'active' state, and unveil the role of the RNA duplex beyond the seed in Ago2.

A two-dimensional replica-exchange molecular dynamics method for simulating RNA folding using sparse experimental restraints.

We present a 2D replica exchange protocol incorporating secondary structure information to dramatically improve 3D RNA folding using molecular dynamics simulations. We show that incorporating base-pairing restraints into all-atom, explicit solvent simulations enables the accurate recapitulation of the global tertiary fold for 4 representative RNAs ranging in length from 24 to 68 nt. This method can potentially utilize base-pairing information from a wide variety of experimental inputs to predict complex RNA tertiary folds including pseudoknots, multi-loop junctions, and non-canonical interactions.

Biodegradation of 1-allyloxy-4-propoxybenzene by selected strains of Pseudomonas putida.

Dialkoxybenzenes constitute a class of organic compounds with anti feeding and oviposition effects on the cabbage looper, Trichoplusia ni. Among them, 1-allyloxy-4-propoxybenzene has the highest feeding deterrence activity and potential for development as commercial insect control agent. To develop this compound, its fate in the environment needs to be studied. The fate of organic compounds in the environment depends on their biodegradability in the soil. We present results of laboratory biodegradation experiments of 1-allyloxy-4-propoxybenzene with three strains of Pseudomonas putida. Two of the three strains of P. putida tested were able to metabolize 1-allyloxy-4-propoxybenzene. Both strains required induction of the catabolic pathway. Specifically, strain ATCC 17453 (which contains the CAM plasmid) metabolized 1-allyloxy-4-propoxybenzene by first dealkylating. This gave both possible monoalkoxy phenols after five days, followed by dihydroquinone after 8 days. In vitro tests with CYP101A1 (cytochrome P450cam, a camphor hydroxylase), revealed that the dealkylation is catalyzed by this enzyme.

Partition, sorption and structure activity relation study of dialkoxybenzenes that modulate insect behavior.

Some dialkoxybenzenes are promising new insect control agents. These compounds mimic naturally occurring odorants that modulate insect behavior. Before applying these compounds, however, their persistence and biodegradability at the application site and in the environment should be understood. The fate of organic compounds in the environment is a complex phenomenon which is influenced by many processes such as sorption to soil components, sedimentation, volatilization, and uptake by plants, as well as biotic and abiotic chemical degradation. In this study, the octanol-water partition coefficient, volatility and sorption on soil components (sand, clay and organic matter) of selected dialkoxybenzenes as well as structure activity relationships with regard to partition, volatility and sorption were investigated. Additionally, calculations of partition, molar volume and molecular surface areas were done, to understand structure-activity relationships of the physical properties.