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Liberibacter asiaticus is the prevalent causative pathogen of Huanglongbing or citrus greening disease, which has resulted in a devastating crisis in the citrus industry. A thorough understanding of this pathogen's physiology and mechanisms to control cell survival is critical in the identification of therapeutic targets. YbeY is a highly conserved bacterial RNase that has been implicated in multiple roles. In this study, we evaluated the biochemical characteristics of the L. asiaticus YbeY (CLIBASIA_01560) and assessed its potential as a target for antimicrobials. YbeY Las was characterized as an endoribonuclease with activity on 3' and 5' termini of 16S and 23S rRNAs, and the capacity to suppress the E. coli ΔybeY phenotype. We predicted the YbeY Las protein:ligand interface and subsequently identified a flavone compound, luteolin, as a selective inhibitor. Site-directed mutagenesis was subsequently used to identify key residues involved in the catalytic activity of YbeY Las. Further evaluation of naturally occurring flavonoids in citrus trees indicated that both flavones and flavonols had potent inhibitory effects on YbeY Las . Luteolin was subsequently examined for efficacy against L. asiaticus in Huanglongbing-infected citrus trees, where a significant reduction in L. asiaticus gene expression was observed., (© 2019 Society for Applied Microbiology and John Wiley & Sons Ltd.)

The RNase H2 complex is a conserved heterotrimeric enzyme that degrades RNA:DNA hybrids and promotes excision of rNMPs misincorporated during DNA replication. Failure to remove ribonucleotides from DNA leads to genomic instability in yeast and humans. The monogenic Aicardi-Goutières syndrome (AGS) results from mutation in one of several genes, among which are those encoding the RNase H2 subunits. The complete cellular and genomic consequences of RNASEH2 mutations and the precise connection to disease remain unclear. To learn more about the effect of RNASEH2 mutations on the cell, we used yeast as a model of AGS disease. We have generated yeast strains bearing AGS-associated mutations in RNASEH2 genes. There is a range of disease presentation in patients bearing these RNASEH2 variants. Here we report on in vivo phenotypes of genomic instability, including mutation and recombination rates, and synthetic gene interactions. These phenotypes provide insight into molecular consequences of RNASEH2 mutations, and lay the groundwork for further study of genomic instability as a contributing factor to AGS disease., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Ribonucleases (RNases) are a large number of enzymes gathered into different bacterial or eukaryotic superfamilies. Bovine pancreatic RNase A, bovine seminal BS-RNase, human pancreatic RNase 1, angiogenin (RNase 5), and amphibian onconase belong to the pancreatic type superfamily, while binase and barnase are in the bacterial RNase N1/T1 family. In physiological conditions, most RNases secreted in the extracellular space counteract the undesired effects of extracellular RNAs and become protective against infections. Instead, if they enter the cell, RNases can digest intracellular RNAs, becoming cytotoxic and having advantageous effects against malignant cells. Their biological activities have been investigated either in vitro , toward a number of different cancer cell lines, or in some cases in vivo to test their potential therapeutic use. However, immunogenicity or other undesired effects have sometimes been associated with their action. Nevertheless, the use of RNases in therapy remains an appealing strategy against some still incurable tumors, such as mesothelioma, melanoma, or pancreatic cancer. The RNase inhibitor (RI) present inside almost all cells is the most efficacious sentry to counteract the ribonucleolytic action against intracellular RNAs because it forms a tight, irreversible and enzymatically inactive complex with many monomeric RNases. Therefore, dimerization or multimerization could represent a useful strategy for RNases to exert a remarkable cytotoxic activity by evading the interaction with RI by steric hindrance. Indeed, the majority of the mentioned RNases can hetero-dimerize with antibody derivatives, or even homo-dimerize or multimerize, spontaneously or artificially. This can occur through weak interactions or upon introducing covalent bonds. Immuno-RNases, in particular, are fusion proteins representing promising drugs by combining high target specificity with easy delivery in tumors. The results concerning the biological features of many RNases reported in the literature are described and discussed in this review. Furthermore, the activities displayed by some RNases forming oligomeric complexes, the mechanisms driving toward these supramolecular structures, and the biological rebounds connected are analyzed. These aspects are offered with the perspective to suggest possible efficacious therapeutic applications for RNases oligomeric derivatives that could contemporarily lack, or strongly reduce, immunogenicity and other undesired side-effects., (Copyright © 2019 Gotte and Menegazzi.)

In this short note we describe the conversion of the widely used fluorescence quenching azo-dyes DABCYL and HABA to fluorophores. The dyes were conjugated to the proteins RNase and human serum albumin (HSA) and subsequently reduced using sodium dithionite (Na 2 S 2 O 4 ), thus forming amine-containing fluorophores. Since this chemical reaction can be applied to any azo-containing quencher compound, a great variety of substances can be readily obtained synthetically. This approach provides a promising tool in the use of fluorescence-based investigations of biomolecular interactions., (Copyright © 2019. Published by Elsevier Inc.)

A powerful computational strategy to determine the equilibrium association constant of two macromolecules with explicit-solvent molecular dynamics (MD) simulations is the "geometric route", which considers the reversible physical separation of the bound complex in solution. Nonetheless, multiple challenges remain to render this type of methodology reliable and computationally efficient in practice. In particular, in one, formulation of the geometric route relies on the potential of mean force (PMF) for physically separating the two binding partners restrained along a straight axis, which must be selected prior to the calculation. However, practical applications indicate that the calculation of the separation PMF along the predefined rectilinear pathway may be suboptimal and slowly convergent. Recognizing that a rectilinear straight separation pathway is generally not representative of how the protein complex physically separates in solution, we put forth a novel theoretical framework for binding free-energy calculations, leaning on the optimal curvilinear minimum free-energy path (MFEP) determined from the string method. The proposed formalism is validated by comparing the results obtained using both rectilinear and curvilinear pathways for a prototypical host-guest complex formed by cucurbit[7]uril (CB[7]) binding benzene, and for the barnase-barstar protein complex. On the basis of multi-microsecond MD calculations, we find that the calculations following the traditional rectilinear pathway and the string-based curvilinear pathway agree quantitatively, but convergence is faster with the latter.

Contact-dependent growth inhibition (CDI) is a form of interbacterial competition mediated by CdiB-CdiA two-partner secretion systems. CdiA effector proteins carry polymorphic C-terminal toxin domains (CdiA-CT), which are neutralized by specific CdiI immunity proteins to prevent self-inhibition. Here, we present the crystal structures of CdiA-CT⋅CdiI complexes from Klebsiella pneumoniae 342 and Escherichia coli 3006. The toxins adopt related folds that resemble the ribonuclease domain of colicin D, and both are isoacceptor-specific tRNases that cleave the acceptor stem of deacylated tRNA GAU Ile . Although the toxins are similar in structure and substrate specificity, CdiA-CT Kp342 activity requires translation factors EF-Tu and EF-Ts, whereas CdiA-CT EC3006 is intrinsically active. Furthermore, the corresponding immunity proteins are unrelated in sequence and structure. CdiI Kp342 forms a dimeric β sandwich, whereas CdiI EC3006 is an α-solenoid monomer. Given that toxin-immunity genes co-evolve as linked pairs, these observations suggest that the similarities in toxin structure and activity reflect functional convergence., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Chromosomally-encoded toxin-antitoxin complexes are ubiquitous in bacteria and regulate growth through the release of the toxin component typically in a stress-dependent manner. Type II ribosome-dependent toxins adopt a RelE-family RNase fold and inhibit translation by degrading mRNAs while bound to the ribosome. Here, we present biochemical and structural studies of the Escherichia coli YoeB toxin interacting with both a UAA stop and an AAU sense codon in pre- and post-mRNA cleavage states to provide insights into possible mRNA substrate selection. Both mRNAs undergo minimal changes during the cleavage event in contrast to type II ribosome-dependent RelE toxin. Further, the 16S rRNA decoding site nucleotides that monitor the mRNA in the aminoacyl(A) site adopt different orientations depending upon which toxin is present. Although YoeB is a RelE family member, it is the sole ribosome-dependent toxin that is dimeric. We show that engineered monomeric YoeB is active against mRNAs bound to both the small and large subunit. However, the stability of monomeric YoeB is reduced ∼20°C, consistent with potential YoeB activation during heat shock in E. coli as previously demonstrated. These data provide a molecular basis for the ability of YoeB to function in response to thermal stress., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Many details of oxidative folding of proteins remain obscure, in particular, the role of oxidized glutathione (GSSG). This study reveals some unknown aspects. When a reduced ribonuclease A refolds in the presence of GSSG, most of its eight cysteines accomplish a very fast glutathionylation. In particular, one single cysteine, identified as Cys95 by mass spectrometry, displays 3600 times higher reactivity when compared with an unperturbed protein cysteine. Furthermore, the other five cysteines show 40-50 times higher reactivity toward GSSG. This phenomenon is partially due to a low p K a value of most of these cysteines (average p K a = 7.9), but the occurrence of a reversible GSSG-ribonuclease complex ( K D = 0.12 mM) is reasonably responsible for the extraordinary hyper-reactivity of Cys95. Neither hyper-reactivity nor some protein-disulfide complexes have been found by reacting a reduced ribonuclease with other natural disulfides i.e., cystine, cystamine, and homocystine. Hyper-reactivity of all cysteines was observed toward 5,5'-dithiobis-(2-nitrobenzoic acid). Given that GSSG is present in high concentrations in the endoplasmic reticulum, this property may shed light on the early step of its oxidative folding. The ultra-rapid glutathionylation of cysteines, only devoted to form disulfides, is a novel property of the molten globule status of the ribonuclease., Competing Interests: The authors declare no conflicts of interest. The funder (University of Rome Tor Vergata) had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

As proteins perform most cellular functions, quantitative understanding of protein energetics is required to gain control of biological phenomena. Accurate models of native proteins can be obtained experimentally, but the lack of equally fine models of unfolded ensembles impedes the calculation of protein folding energetics from first principles. Here, we show that an atomistic unfolded ensemble model, consisting of a few dozen conformations built from a protein sequence, can be used in conjunction with an X-ray structure of its native state to calculate accurately by difference the changes in enthalpy and heat capacity of the polypeptide upon folding. The calculation is done using molecular dynamics simulations, popular force fields, and water models, and for the two model proteins studied (barnase and SNase), the results agree within error or are very close to their experimentally determined properties. The enthalpy sampling of the unfolded ensemble is done through short 2 ns simulations that do not significantly modify the representative distribution of R g of the starting conformations. The impressive accuracy obtained opens the possibility to investigate quantitatively systems or phenomena not amenable to experiment and paves the way for addressing the calculation of protein conformational stability (i.e., the change in Gibbs energy upon folding), a central goal of structural biology. So far, these calculated enthalpy and heat capacity changes, combined with the experimentally determined melting temperatures of the corresponding protein, allow us to reproduce the stability curves of both barnase and SNase.

We described a ribonuclease-dependent cleavable beacon primer, an energy-transfer-tagged oligonucleotide inserted with a ribonucleotide, which can be cleaved by ribonuclease to generate enhanced fluorescence signals and initiate DNA amplification for single nucleotide mutation detection with ultrahigh sensitivity and selectivity.

Ageritin is the first reported ribotoxin-like protein from basidiomycetes fungi. It can induce ribosomal integrity damage and translation block, and interferes with mitochondrial redox activity of some glioma and neuroblastoma cell lines. Herein, Ageritin has been investigated as a valuable neurotoxin towards either undifferentiated or retinoic acid (RA)-differentiated SH-SY5Y neuroblastoma cells showing a selective cell toxicity against undifferentiated cells. MTT and sulforhodamine B (SRB) assays highlighted that Ageritin markedly decreases the mitochondrial redox activity and viability of undifferentiated cells, meanwhile inducing evident morphological changes eliciting neuronal-like appearance in these cells. Data from lactate dehydrogenase release assay, cytofluorimetric analysis and caspase-3 enzymatic activity measurement suggest that Ageritin promotes cell death through a caspase-dependent apoptotic pathway. The Z-VAD-FMK caspase inhibitor was able to prevent this apoptotic pathway activation. Based on the interesting behaviour of Ageritin vs. SH-SY5Y cells, the development of a scale-up procedure to obtain the purified protein in larger amounts (yield 2.5 mg per 100 g) has been optimized.

Fungi produce various defense proteins against antagonists, including ribotoxins. These toxins cleave a single phosphodiester bond within the universally conserved sarcin-ricin loop of ribosomes and inhibit protein biosynthesis. Here, we report on the structure and function of ageritin, a previously reported ribotoxin from the edible mushroom Agrocybe aegerita The amino acid sequence of ageritin was derived from cDNA isolated from the dikaryon A. aegerita AAE-3 and lacks, according to in silico prediction, a signal peptide for classical secretion, predicting a cytoplasmic localization of the protein. The calculated molecular weight of the protein is slightly higher than the one reported for native ageritin. The A. aegerita ageritin-encoding gene, AaeAGT1 , is highly induced during fruiting, and toxicity assays with AaeAGT1 heterologously expressed in Escherichia coli showed a strong toxicity against Aedes aegypti larvae yet not against nematodes. The activity of recombinant A. aegerita ageritin toward rabbit ribosomes was confirmed in vitro Mutagenesis studies revealed a correlation between in vivo and in vitro activities, indicating that entomotoxicity is mediated by ribonucleolytic cleavage. The strong larvicidal activity of ageritin makes this protein a promising candidate for novel biopesticide development. IMPORTANCE Our results suggest a pronounced organismal specificity of a protein toxin with a very conserved intracellular molecular target. The molecular details of the toxin-target interaction will provide important insight into the mechanism of action of protein toxins and the ribosome. This insight might be exploited to develop novel bioinsecticides., (Copyright © 2019 American Society for Microbiology.)

Metabolic networks are webs of integrated reactions organized to maximize growth and replication while minimizing the detrimental impact that reactive metabolites can have on fitness. Enamines and imines, such as 2-aminoacrylate (2AA), are reactive metabolites produced as short-lived intermediates in a number of enzymatic processes. Left unchecked, the inherent reactivity of enamines and imines may perturb the metabolic network. Genetic and biochemical studies have outlined a role for the broadly conserved reactive intermediate deaminase (Rid) (YjgF/YER057c/UK114) protein family, in particular RidA, in catalyzing the hydrolysis of enamines and imines to their ketone product. Herein, we discuss new findings regarding the biological significance of enamine and imine production and outline the importance of RidA in controlling the accumulation of reactive metabolites., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Nucleases are enzymes that can degrade genomic DNA and RNA that decrease the accuracy of quantitative measures of those nucleic acids. Here, we study conventional heating, standard microwave irradiation, and Lyse-It, a microwave-based lysing technology, for the potential to fragment and inactivate DNA and RNA endonucleases. Lyse-It employs the use of highly focused microwave irradiation to the sample ultimately fragmenting and inactivating RNase A, RNase B, and DNase I. Nuclease size and fragmentation were determined visually and quantitatively by SDS polyacrylamide gel electrophoresis and the mini-gel Agilent 2100 Bioanalyzer system, with a weighted size calculated to depict the wide range of nuclease fragmentation. Enzyme activity assays were conducted, and the rates were calculated to determine the effect of various lysing conditions on each of the nucleases. The results shown in this paper clearly demonstrate that Lyse-It is a rapid and highly efficient way to degrade and inactivate nucleases so that nucleic acids can be retained for down-stream detection., Competing Interests: The University of Maryland, Baltimore County (UMBC) has filed and has had issued several patents related to microwave-based lysing and DNA fragmentation. Those patents are licensed to Lyse-It LLC, a Maryland-based biotechnology company, which Professor Geddes currently holds stock. Patent #: US US9500590B2. “Assays for pathogen detection using microwaves for lysing and accelerating metal-enhanced fluorescence.” Patent #: US10294451B2 “Flow and static lysing systems and methods for ultrarapid isolation and fragmentation of biological materials by microwave irradiation”, Chris D. Geddes. Lyse-It LLC was not involved in the study design of the material within this paper, nor do any of the author receive any salaries or consultancies from Lyse-It LLC. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Nuclear spin hyperpolarized water is utilized to obtain protein spectra not only in the folded state but also during the refolding process. Polarization transfer to Ribonuclease Sa through proton exchange and the nuclear Overhauser effect (NOE) results in NMR signal enhancements of amide protons by up to 24-fold. These enhancements enable the measurement of fast two-dimensional NMR spectra on the same time scale as the folding. Resolved amide proton signals corresponding to the folded protein are observed both under folded and refolding conditions, whereby the refolding protein shows smaller transferred signals. Residue-specific evaluation of contributions to the polarization transfer indicates that signals attributed to a relayed intramolecular NOE are not observable in the refolding experiment. These differences are explained by the absence of long-range contacts and faster molecular motions in the unfolded protein. Applications of this method include accessing residue-specific information on structure and dynamics during multistate protein folding.

Candida albicans is a polymorphic fungus responsible for mucosal and skin infections. Candida cells establish themselves into biofilm communities resistant to most currently available antifungal agents. An increase of severe infections ensuing in fungal septic shock in elderly or immunosuppressed patients, along with the emergence of drug-resistant strains, urge the need for the development of alternative antifungal agents. In the search for novel antifungal drugs our laboratory demonstrated that two human ribonucleases from the vertebrate-specific RNaseA superfamily, hRNase3 and hRNase7, display a high anticandidal activity. In a previous work, we proved that the N-terminal region of the RNases was sufficient to reproduce most of the parental protein bactericidal activity. Next, we explored their potency against a fungal pathogen. Here, we have tested the N-terminal derived peptides that correspond to the eight human canonical RNases (RN1-8) against planktonic cells and biofilms of C. albicans . RN3 and RN7 peptides displayed the most potent inhibitory effect with a mechanism of action characterized by cell-wall binding, membrane permeabilization and biofilm eradication activities. Both peptides are able to eradicate planktonic and sessile cells, and to alter their gene expression, reinforcing its role as a lead candidate to develop novel antifungal and antibiofilm therapies.

Target RNA binding to crRNA-bound type III-A CRISPR-Cas multi-subunit Csm surveillance complexes activates cyclic-oligoadenylate (cA n ) formation from ATP subunits positioned within the composite pair of Palm domain pockets of the Csm1 subunit. The generated cA n second messenger in turn targets the CARF domain of trans-acting RNase Csm6, triggering its HEPN domain-based RNase activity. We have undertaken cryo-EM studies on multi-subunit Thermococcus onnurineus Csm effector ternary complexes, as well as X-ray studies on Csm1-Csm4 cassette, both bound to substrate (AMPPNP), intermediates (pppA n ), and products (cA n ), to decipher mechanistic aspects of cA n formation and release. A network of intermolecular hydrogen bond alignments accounts for the observed adenosine specificity, with ligand positioning dictating formation of linear pppA n intermediates and subsequent cA n formation by cyclization. We combine our structural results with published functional studies to highlight mechanistic insights into the role of the Csm effector complex in mediating the cA n signaling pathway., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Type III-A CRISPR-Cas surveillance complexes containing multi-subunit Csm effector, guide, and target RNAs exhibit multiple activities, including formation of cyclic-oligoadenylates (cA n ) from ATP and subsequent cA n -mediated cleavage of single-strand RNA (ssRNA) by the trans-acting Csm6 RNase. Our structure-function studies have focused on Thermococcus onnurineus Csm6 to deduce mechanistic insights into how cA 4 binding to the Csm6 CARF domain triggers the RNase activity of the Csm6 HEPN domain and what factors contribute to regulation of RNA cleavage activity. We demonstrate that the Csm6 CARF domain is a ring nuclease, whereby bound cA 4 is stepwise cleaved initially to ApApApA>p and subsequently to ApA>p in its CARF domain-binding pocket, with such cleavage bursts using a timer mechanism to regulate the RNase activity of the Csm6 HEPN domain. In addition, we establish T. onnurineus Csm6 as an adenosine-specific RNase and identify a histidine in the cA 4 CARF-binding pocket involved in autoinhibitory regulation of RNase activity., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Human noroviruses are the leading cause of viral gastroenteritis. In the absence of a practical culture technique for routine analysis of infectious noroviruses, several methods have been developed to discriminate between infectious and non-infectious viruses by removing non-viable viruses prior to analysis by RT-qPCR. In this study, two such methods (RNase and porcine gastric mucin) which were designed to remove viruses with compromised capsids (and therefore assumed to be non-viable), were assessed for their ability to quantify viable F-specific RNA bacteriophage (FRNAP) and human norovirus following inactivation by UV-C or heat. It was found that while both methods could remove a proportion of non-viable viruses, a large proportion of non-viable virus remained to be detected by RT-qPCR, leading to overestimations of the viable population. A model was then developed to determine the proportion of RT-qPCR detectable RNA from non-viable viruses that must be removed by such methods to reduce overestimation to acceptable levels. In most cases, nearly all non-viable virus must be removed to reduce the log overestimation of viability to within levels that might be considered acceptable (e.g. below 0.5 log 10 ). This model could be applied when developing alternative pre-treatment methods to determine how well they should perform to be comparable to established infectivity assays.

Ribotoxins are fungal proteins that serve as weapons against parasites and insects. They are strongly toxic due to their ability to enter host cells and inactivate ribosomes. Ageritin is the prototype of a new ribotoxin-like protein family present in basidiomycetes. We demonstrate that this enzyme has peculiar binding and enzymatic features. Different from other ribotoxins, its ribonucleolytic activity requires the presence of divalent cations, with a maximum activation in the presence of zinc ions, for which Ageritin exhibits the strongest affinity of binding. We modeled the catalytic metal binding site of Ageritin, made of the putative triad Asp68, Asp70 and His77. This report highlights that Ageritin has the structure and function of an RNase but a Mg 2+ /Zn 2+ -dependent mechanism of action, a new finding for ribotoxins. As a zinc-dependent toxin, Ageritin can be classified among the arsenal of zinc-binding proteins involved in fungal virulence., (Copyright © 2019. Published by Elsevier B.V.)

Ribonuclease (RNase)-mediated degradation of messenger RNA (mRNA) poses a huge obstruction to in vivo mRNA delivery. Herein, we propose a novel strategy to protect mRNA by structuring mRNA to prevent RNase attack through steric hinderance. Bundling of mRNA strands through hybridization of RNA oligonucleotide linkers allowed the preparation of mRNA nano-assemblies (R-NAs) comprised of 7.7 mRNA strands on average, mostly below 100 nm in diameter. R-NA formation boosted RNase stability by around 100-fold compared to naïve mRNA and preserved translational activity, allowing protein production. A mechanistic analysis suggests that an endogenous mRNA unwinding mechanism triggered by 5'-cap-dependent translation may induce selective R-NA dissociation intracellularly, leading to smooth translation. R-NAs showed efficient mRNA transfection in mouse brain, demonstrating the feasibility for in vivo administration., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

An azobenzene-derived photoswitch has been covalently cross-linked to two sites of the S-peptide in the RNase S complex in a manner that the α-helical content of the S-peptide reduces upon cis -to- trans isomerization of the photoswitch. Three complementary experimental techniques have been employed, isothermal titration calorimetry, circular dichroism spectroscopy and intrinsic tyrosine fluorescence quenching, to determine the binding affinity of the S-peptide to the S-protein in the two states of the photoswitch. Five mutants with the photoswitch attached to different sites of the S-peptide have been explored, with the goal to maximize the change in binding affinity upon photoswitching, and to identify the mechanisms that determine the binding affinity. With regard to the first goal, one mutant has been identified, which binds with reasonable affinity in the one state of the photoswitch, while specific binding is completely switched off in the other state. With regard to the second goal, accompanying molecular dynamics simulations combined with a quantitative structure activity relationship revealed that the α-helicity of the S-peptide in the binding pocket correlates surprisingly well with measured dissociation constants. Moreover, the simulations show that both configurations of all S-peptides exhibit quite well-defined structures, even in apparently disordered states.

Metal-dependent DNA and RNA nucleases are enzymes that cleave nucleic acids with great efficiency and precision. These enzyme-mediated hydrolytic reactions are fundamental for the replication, repair, and storage of genetic information within the cell. Here, extensive classical and quantum-based free-energy molecular simulations show that a cation-π interaction is transiently formed in situ at the metal core of Bacteriophage-λ Exonuclease (Exo-λ), during catalysis. This noncovalent interaction (Lys131-Tyr154) triggers nucleophile activation for nucleotide excision. Then, our simulations also show the oscillatory dynamics and swinging of the newly formed cation-π dyad, whose conformational change may favor proton release from the cationic Lys131 to the bulk solution, thus restoring the precatalytic protonation state in Exo-λ. Altogether, we report on the novel mechanistic character of cation-π interactions for catalysis. Structural and bioinformatic analyses support that flexible orientation and transient formation of mobile cation-π interactions may represent a common catalytic strategy to promote nucleic acid hydrolysis in DNA and RNA nucleases.

Key Message: S-RNase was demonstrated to be predominantly recognized by an S locus F-box-like protein and an S haplotype-specific F-box-like protein in compatible pollen tubes of sweet cherry. Self-incompatibility (SI) is a reproductive barrier that rejects self-pollen and inhibits self-fertilization to promote outcrossing. In Solanaceae and Rosaceae, S-RNase-based gametophytic SI (GSI) comprises S-RNase and F-box protein(s) as the pistil and pollen S determinants, respectively. Compatible pollen tubes are assumed to detoxify the internalized cytotoxic S-RNases to maintain growth. S-RNase detoxification is conducted by the Skp1-cullin1-F-box protein complex (SCF) formed by pollen S determinants, S locus F-box proteins (SLFs), in Solanaceae. In Prunus, the general inhibitor (GI), but not pollen S determinant S haplotype-specific F-box protein (SFB), is hypothesized to detoxify S-RNases. Recently, SLF-like proteins 1-3 (SLFL1-3) were suggested as GI candidates, although it is still possible that other proteins function predominantly in GI. To identify the other GI candidates, we isolated four other pollen-expressed SLFL and SFB-like (SFBL) proteins PavSLFL6, PavSLFL7A, PavSFBL1, and PavSFBL2 in sweet cherry. Binding assays with four PavS-RNases indicated that PavSFBL2 bound to PavS 1, 6 -RNase while the others bound to nothing. PavSFBL2 was confirmed to form an SCF complex in vitro. A co-immunoprecipitation assay using the recombinant PavS 6 -RNase as bait against pollen extracts and a mass spectrometry analysis identified the SCF complex components of PavSLFLs and PavSFBL2, M-locus-encoded glutathione S-transferase (MGST), DnaJ-like protein, and other minor proteins. These results suggest that SLFLs and SFBLs could act as predominant GIs in Prunus-specific S-RNase-based GSI.

This work focused on the development of a combined experimental and computational tool set to study protein-mAb interactions. A model protein library was first screened using cross interaction chromatography to identify proteins with the strongest retention. Fluorescence polarization was then employed to study the interactions and thermodynamics of the selected proteins-lactoferrin, pyruvate kinase, and ribonuclease B with the mAb. Binding affinities of lactoferrin and pyruvate kinase to the mAb were seen to be relatively salt insensitive in the range examined. Further, a strong entropic contribution was observed, suggesting the importance of hydrophobic interactions. On the other hand, ribonuclease B-mAb binding was seen to be enthalpically driven and salt sensitive, indicating the importance of electrostatic interactions. Protein-protein docking was then carried out and the results identified the CDR region on the mAb as an important binding site for all three proteins. The binding interfaces identified for the mAb-lactoferrin and mAb-pyruvate kinase systems were found to contain complementary hydrophobic and oppositely charged clusters on the interacting regions which were indicative of both hydrophobic and electrostatic interactions. On the other hand, the binding site on ribonuclease B was predominantly positively charged with minimal hydrophobicity. This resulted in an alignment with negatively charged clusters on the mAb, supporting the contention that these interactions were primarily electrostatic in nature. Importantly, these computational results were found to be consistent with the fluorescence polarization data and this combined approach may have utility in examining mAb-HCP interactions which can often complicate the downstream processing of biologics. © 2019 American Institute of Chemical Engineers., (© 2019 American Institute of Chemical Engineers.)

A chimeric DNA probe-aided RNase H-EXPAR approach is developed for the accurate and specific detection of RNA mutation at single-base resolution through a new site-specific RNase H cleavage mechanism.

Cas13d, the type VI-D CRISPR-Cas effector, is an RNA-guided ribonuclease that has been repurposed to edit RNA in a programmable manner. Here we report the detailed structural and functional analysis of the uncultured Ruminococcus sp. Cas13d (UrCas13d)-crRNA complex. Two hydrated Mg 2+ ions aid in stabilizing the conformation of the crRNA repeat region. Sequestration of divalent metal ions does not alter pre-crRNA processing, but abolishes target cleavage by UrCas13d. Notably, the pre-crRNA processing is executed by the HEPN-2 domain. Furthermore, both the structure and sequence of the nucleotides U(-8)-C(-1) within the repeat region are indispensable for target cleavage, and are specifically recognized by UrCas13d. Moreover, correct base pairings within two separate spacer regions (an internal and a 3'-end region) are essential for target cleavage. These findings provide a framework for the development of Cas13d into a tool for a wide range of applications.

Regnase-1 (also known as Zc3h12a or MCPIP-1) is an endoribonuclease involved in mRNA degradation of inflammation-associated genes. Regnase-1 is inactivated in response to external stimuli through post-translational modifications including phosphorylation, yet the precise role of phosphorylation remains unknown. Here, we demonstrate that interleukin (IL)-17 induces phosphorylation of Regnase-1 in an Act1-TBK1/IKKi-dependent manner, especially in nonhematopoietic cells. Phosphorylated Regnase-1 is released from the endoplasmic reticulum (ER) into the cytosol, thereby losing its mRNA degradation function, which leads to expression of IL-17 target genes. By using CRISPR/Cas-9 technology, we generated Regnase-1 mutant mice, in which IL-17-induced Regnase-1 phosphorylation is completely blocked. Mutant mice ( Regnase-1 AA/AA and Regnase-1 ΔCTD/ΔCTD ) were resistant to the IL-17-mediated inflammation caused by T helper 17 (Th17) cells in vivo. Thus, Regnase-1 plays a critical role in the development of IL-17-mediated inflammatory diseases via the Act1-TBK1-IKKi axis, and blockade of Regnase-1 phosphorylation sites may be promising for treatment of Th17-associated diseases., (© 2019 Tanaka et al.)

Background: The efficiency of RNA interference (RNAi) varies considerably among different insect species, and there is growing evidence to suggest that degradation of double-stranded (dsRNA) prior to uptake is an important factor that limits the efficiency of RNAi in insects. In Locusta migratoria, RNAi is highly efficient when dsRNA is delivered by injection, but not by feeding. However, detailed mechanisms causing such differential RNAi efficiency are still elusive., Results: We identified and characterized the full-length complementary DNAs (cDNAs) of two new dsRNA nuclease (dsRNase) genes from L. migratoria, which were named LmdsRNase1 and LmdsRNase4. Transcript analyses revealed that LmdsRNase1 and LmdsRNase4 were highly expressed in hemolymph with relatively lower expression in other tested tissues. Our study using heterologously expressed LmdsRNase1 and LmdsRNase4 fusion proteins showed that LmdsRNase1 can degrade dsRNA rapidly at an optimal pH of 5, whereas LmdsRNase4 had no activity at any of the pH values examined. In comparing the substrate specificity of the four LmdsRNases, we found that only LmdsRNase1 and LmdsRNase2 digested dsRNA; however, our experiments suggested that the physiological pH of hemolymph (7.0) suppresses LmdsRNase1 activity permitting significant dsRNA stability in this tissue. Conversely, the physiological pH of midgut juice (6.8) is ideal for LmdsRNase2 activity, resulting in degradation of dsRNA in midgut., Conclusion: The physiological pH of different insect tissues or compartments can significantly alter the stability of dsRNA by influencing LmdsRNase activity in L. migratoria. Thus, new strategies to overcome such obstacles are expected to help implement RNAi-based technologies for insect pest management. © 2018 Society of Chemical Industry., (© 2018 Society of Chemical Industry.)

The 3' poly(A) tail of messenger RNA is fundamental to regulating eukaryotic gene expression. Shortening of the poly(A) tail, termed deadenylation, reduces transcript stability and inhibits translation. Nonetheless, the mechanism for poly(A) recognition by the conserved deadenylase complexes Pan2-Pan3 and Ccr4-Not is poorly understood. Here we provide a model for poly(A) RNA recognition by two DEDD-family deadenylase enzymes, Pan2 and the Ccr4-Not nuclease Caf1. Crystal structures of Saccharomyces cerevisiae Pan2 in complex with RNA show that, surprisingly, Pan2 does not form canonical base-specific contacts. Instead, it recognizes the intrinsic stacked, helical conformation of poly(A) RNA. Using a fully reconstituted biochemical system, we show that disruption of this structure-for example, by incorporation of guanosine into poly(A)-inhibits deadenylation by both Pan2 and Caf1. Together, these data establish a paradigm for specific recognition of the conformation of poly(A) RNA by proteins that regulate gene expression.

Recent advances have proven solid-state nanopores as a powerful analysis platform that enables label-free and separation-free single-molecule analysis. However, the relatively low resolution still limits its application because many chemicals or targets with small sizes could not be recognized in a label-free condition. In this paper, we provide a possible solution that uses solid-state nanopores for small species signaling via the transition of huge DNA assembly products. DNAzyme responding to metal ions and the hybridization chain reaction (HCR) generating nanopore-detectable dsDNA concatamers are used as the transition model set. By the two-step DNAzyme-HCR transition, Pb2+ that was too tiny to be sensed was successfully recognized by the nanopore. The whole process happened in a completely homogeneous solution without any chemical modification. During condition optimization, we also discussed one possible application challenge that may affect the HCR signal-background distinction. Solid-state nanopores provide a potential solution to this challenge due to its ability to profile product length or even 3D structure information.

For the improvement of nuclease resistance, four kinds of new modifications through a carbamoylethyl linker were designed. Among them, the 2'-O-[2-N-{2-(benzimidazol-1-yl)ethyl}carbamoylethyl] modification showed 20-fold longer half-life when treated with a 3' to 5' exonuclease compared to the 2'-O-methoxyethyl (MOE) modification, which is used in approved drugs. In addition, this large modification did not disturb the binding affinity or RNase H-dependent antisense activity. From these findings, it could be concluded that an adequate linker, such as carbamoylethyl in this study, could extend the utility of 2'-O-modification without loss of the properties of nucleic acids. This strategy would be useful for the development of nucleic acid therapeutics.

Dynamic self-organisation far from equilibrium is a key concept towards building autonomously acting materials. Here, we report the coupling of an antagonistic enzymatic reaction of RNA polymerisation and degradation to the aggregation of micron sized DNA coated colloids into fractal structures. A transient colloidal aggregation process is controlled by competing reactions of RNA synthesis of linker strands by a RNA polymerase and their degradation by a ribonuclease. By limiting the energy supply (NTP) of the enzymatic reactions, colloidal clusters form and subsequently disintegrate without the need of external stimuli. Here, the autonomous colloidal aggregation and disintegration can be modulated in terms of lifetime and cluster size. By restricting the enzyme activity locally, a directed spatial propagation of a colloidal aggregation and disintegration front is realised.

A DSN-RNAse-TdT-T7 exo probing system allows the detection of miRNA 21 with very high sensitivity (LOD = 2.57 fM) and selectivity-the result of (i) avoiding the false-positive signal from miRNA reacting with TdT polymerase and (ii) signal amplification occurring through a FRET-breaking mechanism involving T7 exo.

The optimization of β-turns has been used as a strategy to increase protein thermal stability. One example is the S48P mutation in Ribonuclease Sa, introduced to optimize a β-turn, which increases the stability of the protein as determined experimentally. Here, we have studied 48 SYGY 51 β-turn and its S48P mutant from RNase Sa, as a peptide and as part of the protein, using molecular dynamics simulations. The turn propensity of the region 48 SYGY 51 shows an increase in both the peptide and protein models on S48P mutation. The mutant protein shows an overall decrease in conformational dynamics and a decrease in conformational heterogeneity as compared to the wildtype protein. A comparatively restricted sampling of the φ-ψ region of GLN47, a pre PRO48 residue, in the mutant protein and some local changes in hydrogen bonding patterns involving residues 20-24 might be contributing to the mutant protein stability. In addition, some long-range hydrogen bonding interactions involving the 60s loop and the salt-bridge interaction involving ASP17-ARG63 could also be contributing to the increase in rigidity and stability of the mutant protein., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Interaction of HIV-1 rev response element (RRE) RNA with its cognate protein, Rev, is critical for HIV-1 replication. Understanding the mode of interaction between RRE RNA and ligands at the binding site can facilitate RNA molecular recognition as well as provide a strategy for developing anti-HIV therapeutics. Our approach utilizes branched peptides as a scaffold for multivalent binding to RRE IIB (high affinity rev binding site) with incorporation of unnatural amino acids to increase affinity via non-canonical interactions with the RNA. Previous high throughput screening of a 46,656-member library revealed several hits that bound RRE IIB RNA in the sub-micromolar range. In particular, the lead compound, 4B3, displayed a K d value of 410 nM and demonstrated selectivity towards RRE. A ribonuclease protection assay revealed that 4B3 binds to the stem-loop structure of RRE IIB RNA, which was confirmed by SHAPE analysis with 234 nt long NL4-3 RRE RNA. Our studies further indicated interaction of 4B3 with both primary and secondary Rev binding sites., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Intrinsically disordered proteins (IDPs) are extensively involved in dynamic signaling processes which require a high association rate and a high dissociation rate for rapid binding/unbinding events and at the same time a sufficient high affinity for specific recognition. Although the coupled folding-binding processes of IDPs have been extensively studied, it is still impossible to predict whether an unfolded protein is suitable for molecular signaling via coupled folding-binding. In this work, we studied the interplay between intrinsic folding mechanisms and coupled folding-binding process for unfolded proteins through molecular dynamics simulations. We first studied the folding process of three representative IDPs with different folded structures, that is, c-Myb, AF9, and E3 rRNase. We found the folding free energy landscapes of IDPs are downhill or show low barriers. To further study the influence of intrinsic folding mechanism on the binding process, we modulated the folding mechanism of barnase via circular permutation and simulated the coupled folding-binding process between unfolded barnase permutant and folded barstar. Although folding of barnase was coupled to target binding, the binding kinetics was significantly affected by the intrinsic folding free energy barrier, where reducing the folding free energy barrier enhances binding rate up to two orders of magnitude. This accelerating effect is different from previous results which reflect the effect of structure flexibility on binding kinetics. Our results suggest that coupling the folding of an unfolded protein with no/low folding free energy barrier with its target binding may provide a way to achieve high specificity and rapid binding/unbinding kinetics simultaneously., (© 2018 Wiley Periodicals, Inc.)

A novel electrochemical biosensor was developed based on multiwall carbon nanotubes/graphene oxide nanoribbons (MWCNTs/GONRs) for sensitive analysis of microRNA-21. Signal-amplified strategy was achieved by duplex-specific nuclease assisted target recycling and alkaline phosphatase-induced redox reactions. At the fabrication process of the sensor, ssDNA capture probes were immobilized on the surface of the MWCNTs@GONRs/AuNPs modified electrode through the Au-S bond, and the streptavidin-conjugated alkaline phosphatase (SA-ALP) was attached to the end of the probe. In the absence of miRNA-21, SA-ALP catalysed the conversion of ascorbic acid 2-phosphate (AAP) into ascorbic acid (AA), triggered a redox reaction under iodine, producing a marked electrochemical response. When miRNA-21 was hybridized to the capture probe, the duplex would be cleaved by the duplex-specific nuclease (DSN), causing the electrochemical signals being significantly decreased as a result of SA-ALP detached from the electrode surface. Under the optimized conditions, our biosensor showed satisfactory sensitivity (detection limit, 0.034 fM), excellent selectivity and good accuracy (recoveries, 77.4-120.2%; RSD, 5.2-7.3%) after systematic evaluations. The proposed approach was applied to detect miRNA-21 from human serum samples, which indicated that it was reliable and could be widely used as an effective tool for rapid detection of the target in serums., (Copyright © 2019 Elsevier B.V. All rights reserved.)

In eukaryotic cells, cytoplasmic mRNA is characterised by a 3' poly(A) tail. The shortening and removal of poly(A) tails (deadenylation) by the Ccr4-Not nuclease complex leads to reduced translational efficiency and RNA degradation. Using recombinant human Caf1 (CNOT7) enzyme as a screening tool, we recently described the discovery and synthesis of a series of substituted 1-hydroxy-3,7-dihydro-1 H -purine-2,6-diones (1-hydroxy-xanthines) as inhibitors of the Caf1 catalytic subunit of the Ccr4-Not complex. Here, we used a chemiluminescence-based AMP detection assay to show that active 1-hydroxy-xanthines inhibit both isolated Caf1 enzyme and human Caf1-containing complexes that also contain the second nuclease subunit Ccr4 (CNOT6L) to a similar extent, indicating that the active site of the Caf1 nuclease subunit does not undergo substantial conformational change when bound to other Ccr4-Not subunits. Using differential scanning fluorimetry, we also show that binding of active 1-hydroxy-xanthines requires the presence of Mg 2+ ions, which are present in the active site of Caf1., Competing Interests: The authors declare no conflict of interest.

Peptide nucleic acid (PNA)-neocuproine conjugates have been shown to efficiently catalyse the cleavage of RNA target sequences in the presence of Cu 2+ ions in a site-specific manner. These artificial enzymes are designed to force the formation of a bulge in the RNA target, the sequence of which has been shown to be key to the catalytic activity. Here, we present a further investigation into the action of Cu 2+ -dependent PNAzymes with respect to the dependence on bulge composition in 3- and 4-nucleotide bulge systems. Cu 2+ -dependent PNAzymes were shown to have a clear preference for 4-nucleotide bulges, as the cleavage of 3-nucleotide bulge-forming RNA sequences was significantly slower, which is illustrated by a shift in the half-lives from approximately 30 min to 24 h. Nonetheless, the nucleotide preferences at different positions in the bulge displayed similar trends in both systems. Moreover, the cleavage site was probed by introducing critical chemical modifications to one of the cleavage site nucleotides of the fastest cleaved 4-nucleotide RNA bulge. Namely, the exclusion of the exocyclic amine of the central adenine and the replacement of the 2'-hydroxyl nucleophile with 2'-H or 2'-OMe substituents in the RNA severely diminished the rate of RNA cleavage by the Cu 2+ -dependent PNAzyme, giving insight into the mechanism of cleavage. Moreover, the shorter recognition arm of the RNA/PNAzyme complex was modified by extending the PNAzyme by two additional nucleobases. The new PNAzyme was able to efficiently promote the cleavage of RNA when fully hybridised to a longer RNA target and even outperform the previous fastest PNAzyme. The improvement was demonstrated in cleavage studies with stoichiometric amounts of either PNAzyme present, and the extended PNAzyme was also shown to give turnover with a 10-fold excess of the RNA target.

Magnesium ions (Mg 2+ ), abundant in living cells, are essential for biomolecular structure, dynamics, and function. The biological importance of Mg 2+ has motivated continuous development and improvement of various Mg 2+ models for molecular dynamics (MD) simulations during the last decades. There are four types of nonbonded Mg 2+ models: the point charge models based on a 12-6 or 12-6-4 type Lennard-Jones (LJ) potential, and the multisite models based on a 12-6 or 12-6-4 LJ potential. Here, we systematically assessed the performance of these four types of nonbonded Mg 2+ models (21 models in total) in terms of maintaining a challenging intermediate state configuration captured in the structure of a prototypical two-metal-ion RNase H complex with an RNA/DNA hybrid. Our data demonstrate that the 12-6-4 multisite models, which account for charge-induced dipole interactions, perform the best in reproducing all the unique coordination modes in this intermediate state and maintaining the correct carboxylate denticity. Our benchmark work provides a useful guideline for MD simulations and structural refinement of Mg 2+ -containing biomolecular systems.

Type V CRISPR-Cas systems are distinguished by a single RNA-guided RuvC domain-containing effector, Cas12. Although effectors of subtypes V-A (Cas12a) and V-B (Cas12b) have been studied in detail, the distinct domain architectures and diverged RuvC sequences of uncharacterized Cas12 proteins suggest unexplored functional diversity. Here, we identify and characterize Cas12c, -g, -h, and -i. Cas12c, -h, and -i demonstrate RNA-guided double-stranded DNA (dsDNA) interference activity. Cas12i exhibits markedly different efficiencies of CRISPR RNA spacer complementary and noncomplementary strand cleavage resulting in predominant dsDNA nicking. Cas12g is an RNA-guided ribonuclease (RNase) with collateral RNase and single-strand DNase activities. Our study reveals the functional diversity emerging along different routes of type V CRISPR-Cas evolution and expands the CRISPR toolbox., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Mutations in certain genes of the Ribonuclease (RNASE) superfamily can cause amyotrophic lateral sclerosis (ALS) through altered RNA processing mechanisms. About 30 of these missense mutations in RNASE5/ANG gene have already been reported in ALS patients. In another gene of the ribonuclease superfamily, ribonuclease 4 (RNASE4), missense mutations and single nucleotide polymorphisms have been identified in patients suffering from ALS. However, their plausible molecular mechanisms of association with ALS are not known. Here, we present the molecular mechanisms of RNASE4 polymorphisms with ALS using all-atom molecular dynamics (MD) simulations followed by functional assay experiments. As most ALS causing mutations in RNASE superfamily proteins affect either the ribonucleolytic or nuclear translocation activity, we examined these functional properties of wild-type and known RNASE4 variants, R10W, A98V, E48D and V75I, using MD simulations. Our simulation predicted that these variants would retain nuclear translocation activity and that E48D would exhibit loss of ribonucleolytic activity, which was subsequently validated by ribonucleolytic assay. Our results give a mechanistic insight into the association of RNASE4 polymorphisms with ALS and show that E48D-RNASE4 would probably be deleterious and cause ALS in individuals harbouring this polymorphism.

RNase He1 is a guanylic acid-specific ribonuclease of the RNase T1 family from Hericium erinaceus (Japanese name: Yamabushitake). Its RNA degrading activity is strongly inhibited by Zn 2+ , similar to other T1 family RNases. However, RNase He1 shows little inhibition of human tumor cell proliferation, unlike RNase Po1, another T1 family RNase from Pleurotus ostreatus (Japanese name: Hiratake). Here, we determined the three-dimensional X-ray crystal structure of RNase He1 in complex with Zn, which revealed that Zn binding most likely prevents substrate entry into the active site due to steric hindrance. This could explain why RNase He1 and other T1 family RNases are inhibited by Zn. The X-ray crystal structures revealed that RNase He1 and RNase Po1 are almost identical in their catalytic sites and in the cysteine residues involved in disulfide bonds that increase their stability. However, our comparison of the electrostatic potentials of their molecular surfaces revealed that RNase He1 is negative whereas RNase Po1 is positive; thus, RNase He1 may not be able to electrostatically bind to the plasma membrane, potentially explaining why it does not exhibit antitumor activity. Hence, we suggest that the cationic characteristics of RNase Po1 are critical to the anti-tumor properties of the protein.

Missense mutations in certain genes of the Ribonuclease (RNASE) superfamily cause amyotrophic lateral sclerosis (ALS) through loss of either ribonucleolytic or nuclear translocation or both of these activities. While rare ANG/RNASE5 variants have been previously shown to be ALS causative, it is not yet known if any of the reported rare RNASE4 variants can also trigger ALS. The study aims to understand whether rare variants of RNASE4 can manifest ALS through similar loss-of-function mechanisms. Molecular dynamics (MD) simulations were performed on wild-type and all reported rare RNASE4 variants to study the structural and dynamic changes in the catalytic triad and nuclear localization signal residues responsible for ribonucleolytic and nuclear translocation activities respectively. Our systematic study comprising a total of 2.1 μs MD simulations reveal that three rare variants M29I, H72P and R95W would lose their ribonucleolytic activity as a result of conformational alteration of catalytic residue His116, and the R31T and R32W variants would lose their nuclear translocation ability due to local folding and reduced solvent accessibility of 30 QRR 32 residues. Our results show that five among the 20 known rare variants in RNASE4 (M29I, H72P, R95W, R31T and R32W) are possibly deleterious and may manifest ALS due to loss-of-function mechanisms. Overall, this is the first study to demonstrate that although rare and not yet clinically correlated, certain rare RNASE4 variants could cause ALS due to their loss-of-function characteristics and highlights the need to discover novel RNASE variants for a comprehensive understanding of structure-function-disease relationships and design effective therapeutics., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Human RNase6 is a small cationic antimicrobial protein that belongs to the vertebrate RNaseA superfamily. All members share a common catalytic mechanism, which involves a conserved catalytic triad, constituted by two histidines and a lysine (His15/His122/Lys38 in RNase6 corresponding to His12/His119/Lys41 in RNaseA). Recently, our first crystal structure of human RNase6 identified an additional His pair (His36/His39) and suggested the presence of a secondary active site., Methods: In this work we have explored RNase6 and RNaseA subsite architecture by X-ray crystallography, site-directed mutagenesis and kinetic characterization., Results: The analysis of two novel crystal structures of RNase6 in complex with phosphate anions at atomic resolution locates a total of nine binding sites and reveals the contribution of Lys87 to phosphate-binding at the secondary active center. Contribution of the second catalytic triad residues to the enzyme activity is confirmed by mutagenesis. RNase6 catalytic site architecture has been compared with an RNaseA engineered variant where a phosphate-binding subsite is converted into a secondary catalytic center (RNaseA-K7H/R10H)., Conclusions: We have identified the residues that participate in RNase6 second catalytic triad (His36/His39/Lys87) and secondary phosphate-binding sites. To note, residues His39 and Lys87 are unique within higher primates. The RNaseA/RNase6 side-by-side comparison correlates the presence of a dual active site in RNase6 with a favored endonuclease-type cleavage pattern., General Significance: An RNase dual catalytic and extended binding site arrangement facilitates the cleavage of polymeric substrates. This is the first report of the presence of two catalytic centers in a single monomer within the RNaseA superfamily., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Hydrogen deuterium exchange mass spectrometry (HDX-MS) has significant potential for protein structure initiatives but its relationship with protein conformations is unclear. We report on the efficacy of HDX-MS to distinguish between native and non-native proteins using a popular approach to calculate HDX protection factors (PFs) from protein structures. The ability of HDX-MS to identify native protein conformations is quantified by binary structural classification such that merits of the approach for protein modelling can be quantified and better understood. We show that highly accurate PF calculations are not a prerequisite for HDX-MS simulations that are capable of effectively discriminating between native and non-native protein folds. The simulations can also be performed directly on unique structures facilitating high-throughput evaluation of many alternate conformations. The ability of HDX-MS to classify the conformations of homo-protein assemblies is also investigated. In contrast to protein monomers, we show a significant lack of correspondence between the simulated and experimental HDX-MS data for these systems with a subsequent decrease in the ability of HDX-MS to identify native states. However, we demonstrate surprisingly high diagnostic ability of the simulated data for assemblies in which a significant proportion of the individual chains occupy protein-protein interfaces. We relate this to the number of peptides that can sample alternate subunit orientations and discuss these observations within the larger context of applying HDX-MS to evaluate protein structures. Graphical Abstract.

Properties of cationic peptides bearing amino or guanidino groups with various side chain lengths that bind to double stranded RNAs (dsRNAs) were investigated. Peptides with shorter side chain lengths effectively bound to dsRNAs (12mers) increasing their thermal stability. NMR measurements suggested that the cationic peptide binds to the inner side of the major groove of dsRNA. These peptides also increased the thermal stability of siRNA and effectively protected from RNase A digestion. On the other hand, both peptides containing amino groups and guanidine groups did not disturb RNAi activity.

Several genes of IFN-mediated pathways in vertebrates, among them the genes that participate in the 2',5'-oligoadenylate synthetase (OAS)/RNase L pathway, have been identified in C. gigas. In the present study, we identified genes, which encode proteins having 2',5'-oligoadenylate degrading activity in C. gigas. These proteins belong to the 2H phosphoesterase superfamily and have sequence similarity to the mammalian A kinase anchoring protein 7 (AKAP7) central domain, which is responsible for the 2',5'-phosphodiesterase (2',5'-PDE) activity. Comparison of the genomic structures of C. gigas proteins with that of AKAP7 suggests that these enzymes originate from a direct common ancestor. However, the identified nucleases are not typical 2',5'-PDEs. The found enzymes catalyse the degradation of 2',5'-linked oligoadenylates in a metal-ion-independent way, yielding products with 2',3' -cyclic phosphate and 5'-OH termini similarly to the 3'-5' bond cleavage in RNA, catalyzed by metal-independent ribonucleases. 3',5'-linked oligoadenylates are not substrates for them. The preferred substrates for the C. gigas enzymes are 5'-triphosphorylated 2',5'-oligoadenylates, whose major cleavage reaction results in the removal of the 5'-triphosphorylated 2',3'-cyclic phosphate derivative, leaving behind the respective unphosphorylated 2',5'-oligoadenylate. Such a cleavage reaction results in the direct inactivation of the biologically active 2-5A molecule. The 2',5'-ribonucleases (2',5'-RNases) from C. gigas could be members of the ancient group of ribonucleases, specific to 2'-5' phosphodiester bond, together with the enzyme that was characterized previously from the marine sponge Tethya aurantium. The novel 2',5'-RNases may play a role in the control of cellular 2-5A levels, thereby limiting damage to host cells after viral infection., (Copyright © 2018. Published by Elsevier B.V.)

Rationale: The functional study and application of an intact glycoprotein require the structural characterization of both the protein backbone and the glycan moiety; the former has been successfully demonstrated with selective fragmentation of the protein backbone in CID and ExD; whether the latter can be achieved with selective fragmentation of the glycan moiety remains to be explored., Methods: RNase B solution was electrosprayed and its intact glycoforms of GlcNAc 2 Man n (n = 5-9) with the highest abundance (charge state z = 16) were isolated individually and fragmented using CID, ETD, HCD, ETciD, and EThcD on the Orbitrap Fusion Lumos Tribrid mass spectrometer; the dissociation parameters were optimized for selective fragmentation of the N-glycan moiety and protein backbone as well as high sequence coverage. The obtained spectra were interpreted using the protein and N-glycan database search engines ProteinGoggle and GlySeeker, respectively., Results: With exploration of different dissociation parameters for all the five methods, selective fragmentation of the N-glycan moiety (the protein backbone staying intact) was observed in both HCD and EThcD at low collisional energies, but only a few matched product ions were observed; more comprehensive fragmentation was observed at high collisional energies (the protein backbone lost). Selective protein backbone fragmentation was observed in all the five dissociation methods., Conclusions: For comprehensive structural characterization of intact N-glycoproteins using tandem mass spectrometry, the composition and topology of the N-glycan moiety can be identified using HCD and EThcD complementarily at low and high energies; while the amino acid sequence and glycosite can be identified using CID, ETD, HCD, ETciD, and EThcD with their optimal dissociation parameters., (© 2018 John Wiley & Sons, Ltd.)