The Significance of the DUF283 Domain for the Activity of Human Ribonuclease Dicer

Dicers are multidomain proteins, usually comprising an amino-terminal putative helicase domain, a DUF283 domain (domain of unknown function), a PAZ domain, two RNase III domains (RNase IIIa and RNase IIIb) and a dsRNA-binding domain. Dicer homologs play an important role in the biogenesis of small regulatory RNAs by cleaving single-stranded precursors adopting stem-loop structures (pre-miRNAs) and double-strand RNAs into short RNA duplexes containing functional microRNAs or small interfering RNAs, respectively. Growing evidence shows that apart from the canonical role, Dicer proteins can serve a number of other functions. For example, results of our previous studies showed that human Dicer (hDicer), presumably through its DUF283 domain, can facilitate hybridization between two complementary RNAs, thus, acting as a nucleic acid annealer. Here, to test this assumption, we prepared a hDicer deletion variant lacking the amino acid residues 625-752 corresponding to the DUF283 domain. The respective 128-amino acid fragment of hDicer was earlier demonstrated to accelerate base-pairing between two complementary RNAs in vitro. We show that the ΔDUF(625-752) hDicer variant loses the potential to facilitate RNA-RNA base pairing, which strongly proves our hypothesis about the importance of the DUF283 domain for the RNA-RNA annealing activity of hDicer. Interestingly, the in vitro biochemical characterization of the obtained deletion variant reveals that it displays different RNA cleavage properties depending on the pre-miRNA substrate.     

An Additional Insight into the Correlation between the Distribution Ratios and the Aqueous Acidity of the TODGA System

Abstract

Extraction of Eu(III) and Am(III) from HNO₃ into the organic solvents using N,N,N′,N′‐tetraoctyl‐diglycolamide (TODGA) was investigated in order to study the detailed extraction reaction. The chemical species: 1:2 for metal:TODGA complex is present in polar diluents. On the other hand, the metal complexes need three or more TODGA molecules to remain stable in non‐polar diluents. The HNO₃ concentration dependence on the distribution ratio suggests that HNO₃ participates in the metal extraction. Infrared spectra indicate that the carbonyl oxygen coordinates with Eu(III), and luminescence lifetimes suggest that there are no water molecules in the inner coordination sphere of the extracted Eu‐complex.     

Insight into the Structure and Activity of Surface-Engineered Lipase Biofluids

Despite a successful application of solvent-free liquid protein (biofluids) concept to a number of commercial enzymes, the technical advantages of enzyme biofluids as hyperthermal stable biocatalysts cannot be fully utilized as up to 90–99% of native activities are lost when enzymes were made into biofluids. With a two-step strategy (site-directed mutagenesis and synthesis of variant biofluids) on Bacillus subtilis lipase A (BsLA), we elucidated a strong dependency of structure and activity on the number and distribution of polymer surfactant binding sites on BsLA surface. Here, it is demonstrated that improved BsLA variants can be engineered via site-mutagenesis by a rational design, either with enhanced activity in aqueous solution in native form, or with improved physical property and increased activity in solvent-free system in the form of a protein liquid. This work answered some fundamental questions about the surface characteristics for construction of biofluids, useful for identifying new strategies for developing advantageous biocatalysts.     

Natural Mutations Affect Structure and Function of gC1q Domain of Otolin-1

Otolin-1 is a scaffold protein of otoliths and otoconia, calcium carbonate biominerals from the inner ear. It contains a gC1q domain responsible for trimerization and binding of Ca²⁺. Knowledge of a structure–function relationship of gC1q domain of otolin-1 is crucial for understanding the biology of balance sensing. Here, we show how natural variants alter the structure of gC1q otolin-1 and how Ca²⁺ are able to revert some effects of the mutations. We discovered that natural substitutions: R339S, R342W and R402P negatively affect the stability of apo-gC1q otolin-1, and that Q426R has a stabilizing effect. In the presence of Ca²⁺, R342W and Q426R were stabilized at higher Ca²⁺ concentrations than the wild-type form, and R402P was completely insensitive to Ca²⁺. The mutations affected the self-association of gC1q otolin-1 by inducing detrimental aggregation (R342W) or disabling the trimerization (R402P) of the protein. Our results indicate that the natural variants of gC1q otolin-1 may have a potential to cause pathological changes in otoconia and otoconial membrane, which could affect sensing of balance and increase the probability of occurrence of benign paroxysmal positional vertigo (BPPV).     

Thermodynamic Insight into the Effects of Water Displacement and Rearrangement upon Ligand Modifications using Molecular Dynamics Simulations

Computational methods, namely molecular dynamics (MD) simulations in combination with inhomogeneous fluid solvation theory (IFST) were used to retrospectively investigate various cases of ligand structure modifications that led to the displacement of binding site water molecules. Our findings are that water displacement per se is energetically unfavorable in the discussed examples, and that it is merely the fine balance between change in protein–ligand interaction energy, ligand solvation free energies, and binding site solvation free energies that determine if water displacement is favorable or not. We furthermore evaluated if we can reproduce experimental binding affinities by a computational approach combining changes in solvation free energies with changes in protein–ligand interaction energies and entropies. In two of the seven cases, this estimation led to large errors, implying that accurate predictions of relative binding free energies based on solvent thermodynamics is challenging. Nevertheless, MD simulations can provide insight regarding which water molecules can be targeted for displacement.     

Cold-Induced Physiological and Biochemical Alternations and Proteomic Insight into the Response of Saccharum spontaneum to Low Temperature

Sugarcane, a cash crop, is easily affected by low temperature, which results in a decrease in yield and sugar production. Breeding a new variety with cold tolerance is an essential strategy to reduce loss from cold stress. The identification of germplasms and genes/proteins with cold tolerance is a vital step in breeding sugarcane varieties with cold tolerance via a conventional program and molecular technology. In this study, the physiological and biochemical indices of 22 genotypes of S. spontaneum were measured, and the membership function analysis method was used to comprehensively evaluate the cold tolerance ability of these genotypes. The physiological and biochemical indices of these S. spontaneum genotypes showed a sophisticated response to low temperature. On the basis of the physiological and chemical indices, the genotypes were classified into different cold tolerance groups. Then, the high-tolerance genotype 1027 and the low-tolerance genotype 3217 were selected for DIA-based proteomic analysis by subjecting them to low temperature. From the four comparison groups, 1123, 1341, 751, and 1693 differentially abundant proteins (DAPs) were identified, respectively. The DAPs based on genotypes or treatments participated in distinct metabolic pathways. Through detailed analysis of the DAPs, some proteins related to protein homeostasis, carbohydrate and energy metabolism, amino acid transport and metabolism, signal transduction, and the cytoskeleton may be involved in sugarcane tolerance to cold stress. Furthermore, five important proteins related to cold tolerance were discovered for the first time in this study. This work not only provides the germplasms and candidate target proteins for breeding sugarcane varieties with cold tolerance via a conventional program and molecular breeding, but also helps to accelerate the determination of the molecular mechanism underlying cold tolerance in sugarcane.     

Biochemical and Structural Insights into the Winged Helix Domain of P150, the Largest Subunit of the Chromatin Assembly Factor 1

The Chromatin Assembly Factor 1 is a heterotrimeric complex responsible for the nucleosome assembly during DNA replication and DNA repair. In humans, the largest subunit P150 is the major actor of this process. It has been recently considered as a tumor-associated protein due to its overexpression in many malignancies. Structural and functional studies targeting P150 are still limited and only scarce information about this subunit is currently available. Literature data and bioinformatics analysis assisted the identification of a stable DNA binding domain, encompassing residues from 721 to 860 of P150 within the full-length protein. This domain was recombinantly produced and in vitro investigated. An acidic region modulating its DNA binding ability was also identified and characterized. Results showed similarities and differences between the P150 and its yeast homologue, namely Cac-1, suggesting that, although sharing a common biological function, the two proteins may also possess different features.     

Mechanistic Aspects of hSOD1 Maturation from the Solution Structure of CuI‐Loaded hCCS Domain 1 and Analysis of Disulfide‐Free hSOD1 Mutants

Superoxide dismutase 1 (SOD1) maturation within the cell is mainly accomplished with the SOD1‐specific chaperone, CCS, a dimeric protein with three distinct domains in each monomer. We recently showed that the first domain of human CCS (hCCSD1) is responsible for copper transfer to its protein partner, human SOD1 (hSOD1). The NMR solution structure of the copper(I)‐loaded form of hCCSD1 reported here contributes further to characterization of the copper‐transfer mechanism to hSOD1. NMR spectroscopy was also used to examine the hSOD1 mutants C57A, C146A, and C57A/C146A, which are unable to form the structurally conserved disulfide bond in SOD1, in order to investigate the role of these cysteines during hSOD1 copper acquisition. Together, the information on both hCCS and hSOD1, along with a sequence analysis of eukaryotic CCSD1, allows us to propose important mechanistic aspects regarding the copper‐transfer process from hCCS to hSOD1.     

Genome Study of a Novel Virulent Phage vB_SspS_KASIA and Mu-like Prophages of Shewanella sp. M16 Provides Insights into the Genetic Diversity of the Shewanella Virome

Shewanella is a ubiquitous bacterial genus of aquatic ecosystems, and its bacteriophages are also isolated from aquatic environments (oceans, lakes, ice, and wastewater). In this study, the isolation and characterization of a novel virulent Shewanella phage vB_SspS_KASIA and the identification of three prophages of its host, Shewanella sp. M16, including a mitomycin-inducible Mu-like siphovirus, vB_SspS_MuM16-1, became the starting point for comparative analyses of phages infecting Shewanella spp. and the determination of their position among the known bacterial viruses. A similarity networking analysis revealed the high diversity of Shewanella phages in general, with vB_SspS_KASIA clustering exclusively with Colwellia phage 9A, with which it forms a single viral cluster composed of two separate viral subclusters. Furthermore, vB_SspS_MuM16-1 presented itself as being significantly different from the phages deposited in public databases, expanding the diversity of the known Mu-like phages and giving potential molecular markers for the identification of Mu-like prophages in bacterial genomes. Moreover, the functional analysis performed for vB_SspS_KASIA suggested that, despite the KASIA host, the M16 strain grows better in a rich medium and at 30 °C the phage replication cycle seems to be optimal in restrictive culture conditions mimicking their natural environment, the Zloty Stok gold and arsenic mine.