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Background The role of ribonucleases in tuberculosis among people with human immunodeficiency virus (HIV; PWH) is unknown. We explored ribonuclease activity in plasma from PWH with and without tuberculosis. Methods Participants were identified from a cohort of treatment-naive PWH in Ethiopia who had been classified for tuberculosis disease (HIV positive [HIV+]/tuberculosis positive [tuberculosis+] or HIV+/tuberculosis negative [tuberculosis−]). Ribonuclease activity in plasma was investigated by quantification of synthetic spike-in RNAs using sequencing and quantitative polymerase chain reaction and by a specific ribonuclease activity assay. Quantification of ribonuclease 1, 2, 3, 6, 7, and T2 proteins was performed by enzyme-linked immunosorbent assay. Ribonuclease activity and protein concentrations were correlated with markers of tuberculosis and HIV disease severity and with concentrations of inflammatory mediators. Results Ribonuclease activity was significantly higher in plasma of HIV+/tuberculosis+ (n = 51) compared with HIV+/tuberculosis− (n = 78), causing reduced stability of synthetic spike-in RNAs. Concentrations of ribonucleases 2, 3, and T2 were also significantly increased in HIV+/tuberculosis+ compared with HIV+/tuberculosis−. Ribonuclease activity was correlated with HIV viral load, and inversely correlated with CD4 cell count, mid–upper arm circumference, and body mass index. Moreover, ribonuclease activity was correlated with concentrations of interleukin 27, procalcitonin and the kynurenine-tryptophan ratio. Conclusions PWH with tuberculosis disease have elevated plasma ribonuclease activity, which is also associated with HIV disease severity and systemic inflammation. [ABSTRACT FROM AUTHOR]

Toxin-antitoxin (TA) systems are commonly found in most species of bacteria, including the causative agent of tuberculosis M. tuberculosis. TA systems play a role in the adaptation of the pathogen to stresses during host infection. Here we present the results of our study of the effect of the mutation in the vapC46 (C113G) gene using Mycolicibacterium smegmatis as a model. Expression of wild-type and mutant variants of the vapC46 gene in pKW08-MCS-Int vector was shown to increase resistance to erythromycin as well as the survival of M. smegmatis cells under short-term exposure to 0.5 mM H2O2. The introduction of the wild-type variant of vapC46 gene into M. smegmatis cells leads to a slowdown in growth. Expression of the mutant variant of the vapC46 gene did not affect the growth rate. However, no change in the level of RNase activity in cells carrying the vapC46 C113G mutation was observed in an in vitro model. Thus, the VapC46 toxin is involved in the regulation of the survival of M. smegmatis under oxidative stress, and the C113G mutation does not affect this function. The normal growth rate of M. smegmatis, containing the mutant vapC46 gene, along with the absence of changes in the ribonuclease activity of the VapC46 toxin, may be associated either with a change in the specificity of the mutant toxin for bacterial cell mRNA, or with the use of tRNA or rRNA as a substrate. The study of the effects of introducing a mutation into the vapC46 gene on the specificity of the VapC46 toxin is the subject of our further research. [ABSTRACT FROM AUTHOR]

The VapBC system, which belongs to the type II toxin–antitoxin (TA) system, is the most abundant and widely studied system in Mycobacterium tuberculosis. The VapB antitoxin suppresses the activity of the VapC toxin through a stable protein–protein complex. However, under environmental stress, the balance between toxin and antitoxin is disrupted, leading to the release of free toxin and bacteriostatic state. This study introduces the Rv0229c, a putative VapC51 toxin, and aims to provide a better understanding of its discovered function. The structure of the Rv0229c shows a typical PIN-domain protein, exhibiting an β1-α1-α2-β2-α3-α4-β3-α5-α6-β4-α7-β5 topology. The structure-based sequence alignment showed four electronegative residues in the active site of Rv0229c, which is composed of Asp8, Glu42, Asp95, and Asp113. By comparing the active site with existing VapC proteins, we have demonstrated the justification for naming it VapC51 at the molecular level. In an in vitro ribonuclease activity assay, Rv0229c showed ribonuclease activity dependent on the concentration of metal ions such as Mg2+ and Mn2+. In addition, magnesium was found to have a greater effect on VapC51 activity than manganese. Through these structural and experimental studies, we provide evidence for the functional role of Rv0229c as a VapC51 toxin. Overall, this study aims to enhance our understanding of the VapBC system in M. tuberculosis. [ABSTRACT FROM AUTHOR]

Exoribonucleases are frequently used as nuclei acids detection tools for their sequences, modifications, and structures. Escherichia coli ribonuclease R (EcR) is the prototypical exoribonuclease of the RNase II/RNB family degrading RNA in the 3'–5' direction. Different from RNase II, EcR is capable of degrading structured RNA efficiently, which makes it a potential analysis tool for various RNA species. In this work, we examined the nuclease activity of EcR degrading a series of RNA substrates with various sequences. Our biochemical work reveals that EcR is significantly sensitive to cytosine compared with other bases when catalyzing RNA degradation. EcR shows higher cytosine sensitivity compared to its homolog RNase II when degrading RNAs, and the hydrolysis process of EcR is transiently halted and produces apparent intermediate product when the 1-nt upstream of C is A or U, or G. Furthermore, the substitution of glycine with proline (G273P) in EcR enhances its cytosine sensitivity. These findings expand our understanding of EcR enzymatic activities. The EcR G273P mutant bearing higher cytosine sensitivity could help enrich cytosine trails in RNAs and will have potential implications in the detection and analysis of various RNA species especially small RNAs in biological and clinical samples. [ABSTRACT FROM AUTHOR]

Meloidogyne arenaria belongs to root-knot nematodes (RKNs) which constitute a group of highly polyphagous nematodes causing serious damages to many crop varieties. Maize (Zea mays) is one of its main hosts. During plant response to RKN infection, many mechanisms are involved. Pathogenesis-related proteins (PRs), which present many functions and en- zymatic activities, such as ribonucleases (RNases), antioxidative enzymes, or proteases are involved in these processes. The aim of this study was to describe changes in peroxidase and RNase activities induced in Z. mays during its response to M. arenaria infection. Moreover, proteins potentially responsible for peroxidase activity were indicated. RNase and peroxi- dase activities were tested on proteins extracted from roots of healthy plants, M. arenaria infected plants, and healthy plants mixed with M. arenaria juveniles, in native polyacryla- mide (PAA) gels. Samples were collected from two varieties of maize at four time points. A selected fraction showing peroxidase activity was excised from the gel and analyzed using mass spectrometry (MS) to determine protein factors responsible for enzymatic activity. As a result, the analyzed varieties showed slight differences in their RNase and peroxidase activities. Higher activity was observed in the Tasty Sweet variety than in the Waza variety. There were no significant differences between healthy and infected plants in RNase activi- ties at all time points. This was in contrast to peroxidase activity, which was the highest in M. arenaria-infected plants 15 days after inoculation. On the basis of protein identification in excised gel fractions using MS it can be assumed that mainly peroxidase 12 is responsible for the observed peroxidase activity. Moreover, peroxidase activity may be presented by glutathione-S-transferase as well.

Bacterial toxin–antitoxin (TA) systems correlate strongly with physiological processes in bacteria, such as growth arrest, survival and apoptosis. Here, the first crystal structure of a type II TA complex structure of Klebsiella pneumoniae at 2.3 Å resolution is presented. The K. pneumoniae MazEF complex consists of two MazEs and four MazFs in a heterohexameric assembly. It was estimated that MazEF forms a dodecamer with two heterohexameric MazEF complexes in solution, and a truncated complex exists in heterohexameric form. The MazE antitoxin interacts with the MazF toxin via two binding modes, namely, hydrophobic and hydrophilic interactions. Compared with structural homologs, K. pneumoniae MazF shows distinct features in loops β1–β2, β3–β4 and β4–β5. It can be inferred that these three loops have the potential to represent the unique characteristics of MazF, especially various substrate recognition sites. In addition, K. pneumoniae MazF shows ribonuclease activity and the catalytic core of MazF lies in an RNA-binding pocket. Mutation experiments and cell-growth assays confirm Arg28 and Thr51 as critical residues for MazF ribonuclease activity. The findings shown here may contribute to the understanding of the bacterial MazEF TA system and the exploration of antimicrobial candidates to treat drug-resistant K. pneumoniae.

The VapBC system, which belongs to the type II toxin–antitoxin (TA) system, is the most abundant and widely studied system in Mycobacterium tuberculosis. The VapB antitoxin suppresses the activity of the VapC toxin through a stable protein–protein complex. However, under environmental stress, the balance between toxin and antitoxin is disrupted, leading to the release of free toxin and bacteriostatic state. This study introduces the Rv0229c, a putative VapC51 toxin, and aims to provide a better understanding of its discovered function. The structure of the Rv0229c shows a typical PIN-domain protein, exhibiting an β1-α1-α2-β2-α3-α4-β3-α5-α6-β4-α7-β5 topology. The structure-based sequence alignment showed four electronegative residues in the active site of Rv0229c, which is composed of Asp8, Glu42, Asp95, and Asp113. By comparing the active site with existing VapC proteins, we have demonstrated the justification for naming it VapC51 at the molecular level. In an in vitro ribonuclease activity assay, Rv0229c showed ribonuclease activity dependent on the concentration of metal ions such as Mg2+ and Mn2+. In addition, magnesium was found to have a greater effect on VapC51 activity than manganese. Through these structural and experimental studies, we provide evidence for the functional role of Rv0229c as a VapC51 toxin. Overall, this study aims to enhance our understanding of the VapBC system in M. tuberculosis.

Meloidogyne arenaria belongs to root-knot nematodes (RKNs) which constitute a group of highly polyphagous nematodes causing serious damages to many crop varieties. Maize (Zea mays) is one of its main hosts. During plant response to RKN infection, many mechanisms are involved. Pathogenesis-related proteins (PRs), which present many functions and enzymatic activities, such as ribonucleases (RNases), antioxidative enzymes, or proteases are involved in these processes. The aim of this study was to describe changes in peroxidase and RNase activities induced in Z. mays during its response to M. arenaria infection. Moreover, proteins potentially responsible for peroxidase activity were indicated. RNase and peroxidase activities were tested on proteins extracted from roots of healthy plants, M. arenaria infected plants, and healthy plants mixed with M. arenaria juveniles, in native polyacrylamide (PAA) gels. Samples were collected from two varieties of maize at four time points. A selected fraction showing peroxidase activity was excised from the gel and analyzed using mass spectrometry (MS) to determine protein factors responsible for enzymatic activity. As a result, the analyzed varieties showed slight differences in their RNase and peroxidase activities. Higher activity was observed in the Tasty Sweet variety than in the Waza variety. There were no significant differences between healthy and infected plants in RNase activities at all time points. This was in contrast to peroxidase activity, which was the highest in M. arenaria-infected plants 15 days after inoculation. On the basis of protein identification in excised gel fractions using MS it can be assumed that mainly peroxidase 12 is responsible for the observed peroxidase activity. Moreover, peroxidase activity may be presented by glutathione-S-transferase as well. [ABSTRACT FROM AUTHOR]

To replicate efficiently and evade the antiviral immune response of the host, some viruses degrade host mRNA to induce host gene shutoff via encoding shutoff factors. In this study, we found that feline calicivirus (FCV) infection promotes the degradation of endogenous and exogenous mRNAs and induces host gene shutoff, which results in global inhibition of host protein synthesis. Screening assays revealed that proteinase-polymerase (PP) is a most effective factor in reducing mRNA expression. Moreover, PP from differently virulent strains of FCV could induce mRNA degradation. Further, we found that the key sites of the PP protein required for its proteinase activity are also essential for its shutoff activity but also required for viral replication. The mechanism analysis showed that PP mainly targets Pol IItranscribed RNA in a ribosome-, 59 cap-, and 39 poly(A) tail-independent manner. Moreover, purified glutathione S-transferase (GST)-PP fusion protein exhibits RNase activity in vitro in assays using green fluorescent protein (GFP) RNA transcribed in vitro as a substrate in the absence of other viral or cellular proteins. Finally, PPinduced shutoff requires host Xrn1 to complete further RNA degradation. This study provides a newly discovered strategy in which FCV PP protein induces host gene shutoff by promoting the degradation of host mRNAs. IMPORTANCE Virus infection-induced shutoff is the result of targeted or global manipulation of cellular gene expression and leads to efficient viral replication and immune evasion. FCV is a highly contagious pathogen that persistently infects cats. It is unknown how FCV blocks the host immune response and persistently exists in cats. In this study, we found that FCV infection promotes the degradation of host mRNAs and induces host gene shutoff via a common strategy. Further, PP protein for different FCV strains is a key factor that enhances mRNA degradation. An in vitro assay showed that the GST-PP fusion protein possesses RNase activity in the absence of other viral or cellular proteins. This study demonstrates that FCV induces host gene shutoff by promoting the degradation of host mRNAs, thereby introducing a potential mechanism by which FCV infection inhibits the immune response. [ABSTRACT FROM AUTHOR]

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 Mg2+/Zn2+-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. [ABSTRACT FROM AUTHOR]

Abstract Ageritin has been recently described as the first ribotoxin-like from Basidiomycota division (mushroom Agrocybe aegerita) with known antitumor activity (BBA 2017, 1861: 1113-1121). By investigating structural, catalytic and binding properties, we demonstrate that Ageritin is a unique ribotoxin-like protein. Indeed, typical of the ribotoxin family, it shows the specific ribonucleolytic activity against the ribosomal Sarcin-Ricin Loop in a rabbit reticulocytes assay. However, it displays several elements of novelty, as this activity is strongly metal-dependent and completely suppressed in the presence of EDTA, different from other representative members of the ribotoxin family. Consistently, we prove that Ageritin is able to bind magnesium ions with low micromolar affinity. We also show that Ageritin is significantly more stable than other ribotoxins in thermal and chemical denaturation experiments. These peculiar features make Ageritin the prototype of a new ribotoxin-like family present in basidiomycetes. Finally, given its high stability, this enzyme is a promising candidate as a new tool in immunoconjugates and nanoconstructs. Graphical abstract Unlabelled Image Highlights • Ribonucleolytic activity of Ageritin against the ribosome is suppressed by EDTA. • Ageritin is a novel metal-binding ribotoxin. • Ageritin is the most stable ribotoxin so far reported. [ABSTRACT FROM AUTHOR]

In this study, two pathogenesis-related (PR) class 10 protein isoforms, ASPR-1 and ASPR-2, were purified from fresh roots of the Chinese medicinal plant Angelica sinensis (A. sinensis) using 80% ammonium sulfate precipitation, Sephadex G50 gel filtration chromatography, and DEAE-Sepharose ion-exchange chromatography. The molecular masses of ASPR-1 and ASPR-2 were estimated to be 16.66 kDa and 16.46 kDa, respectively, using sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The isoforms are both glycoproteins containing glycosyl contents of 1.8% (ASPR-1) and 3.4% (ASPR-2). The two isoforms were predominantly present as monomers, but they partially dimerized in solution. The 15 N-terminal amino acids of ASPR-1 were determined to be GIQKTEVEAPSTVSA, with significant sequence homology to certain PR-10 proteins. ASPR-2 was also identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) analysis to be a PR-10 protein. The isoforms both exhibited ribonuclease (RNase) activity, with ASPR-2 having higher specific activity (128.85 U mg −1 ) than ASPR-1 (68.67 U mg −1 ). The isoforms had the same optimal temperature of 50 °C but different optimal pH values of 5.0 (ASPR-1) and 6.0 (ASPR-2). The RNase activities of the isoforms were both stable for 30 min at 50 °C, rapidly decreasing at higher or lower processing temperatures. However, ASPR-1 retained higher residual activity (89.4%–80.9%) than ASPR-2 (74.3%–67.9%) at temperatures from 40 °C to 60 °C. These results provide additional information to enrich the current knowledge of poorly annotated A. sinensis proteins. [ABSTRACT FROM AUTHOR]

Shigella flexneri is a Gram-negative anaerobic bacterium that causes highly infectious bacterial dysentery in humans. Here, we solved the crystal structure of SF216, a hypothetical protein from the S. flexneri 5a strain M90T, at 1.7 Å resolution. The crystal structure of SF216 represents a homotrimer stabilized by intersubunit interactions and ion-mediated electrostatic interactions. Each subunit consists of three β-strands and five α-helices with the β-β-β-α-α-α-α-α topology. Based on the structural information, we also demonstrate that SF216 shows weak ribonuclease activity by a fluorescence quenching assay. Furthermore, we identify potential druggable pockets (putative hot spots) on the surface of the SF216 structure by computational mapping. [ABSTRACT FROM AUTHOR]

Background Several species belonging to Ascomycota phylum produce extracellular ribonucleases, known as ribotoxins, which exhibit RNase activity through the cleavage of a single phosphodiester bond, located at the universally conserved sarcin/ricin loop of the large rRNA leading to inhibition of protein biosynthesis. Clarifying the structure-function relationship in ribotoxins is interesting for their use in human tumour therapy and in construction of pest resistant transgenic plants. Results The ribotoxin Ageritin has been isolated for the first time from the Basidiomycetes class. The enzyme, characterized by means of its amino acid composition, N-terminal sequence and a circular dichroism, structurally differs from Ascomycota ribotoxin prototype, although it was able, as α-sarcin, to release a specific α-fragment. However, it does not display aspecific ribonucleolytic activity. Ageritin exerts cytotoxicity and cell death promoting effects towards CNS model cell lines (SK-N-BE(2)-C, U-251 and C6), as vinblastine, a plant alkaloid used in cancer therapy. Moreover, our results indicate that Ageritin initially activates caspase-8, whereas caspase-9 cleavage was not detected, demonstrating the involvement of an extrinsic apoptotic pathway. Conclusions Our findings show that Ageritin is the earliest diverging member of the Ascomycota ribotoxin family, suggesting that ribotoxins are more widely distributed among fungi than previously believed. General significance Ageritin, structurally different from the widely known Ascomycota ribotoxins, with promising anti-cancer properties vs. aggressive brain tumours, has been found from the basidiomycete fungus Agrocybe aegerita . Finally, this finding highlights that the ribotoxin family has divergent members in Basidiomycota phylum, whose structural and functional characterization can give new information on ribotoxin or ribonuclease superfamilies. [ABSTRACT FROM AUTHOR]

Artificial ribonucleases (aRNases) are small compounds catalysing RNA cleavage. Recently we demonstrated that aRNases readily inactivate various viruses in vitro . Here, for three series of aRNases (1,4-diazabicyclo [2.2.2]octane-based and peptide-like compounds) we show that apart from ribonuclease activity the aRNases display chaotropic-like and membranolytic activities. The levels of membranolytic and chaotropic-like activities correlate well with the efficiency of various viruses inactivation (enveloped, non-enveloped, RNA-, DNA-containing). We evaluated the impact of these activities on the efficiency of virus inactivation and found: i) the synergism between membranolytic and chaotropic-like activities is sufficient for the inactivation of enveloped viruses (influenza A, encephalitis, vaccinia viruses) for 1,4-diazabicyclo [2.2.2]octane based aRNases, ii) the inactivation of non-enveloped viruses (encephalomyocarditis, acute bee paralysis viruses) is totally dependent on the synergism of chaotropic-like and ribonuclease activities, iii) ribonuclease activity plays a leading role in the inactivation of RNA viruses by aRNases Dp12F6, Dtr12 and K-D-1, iv) peptide-like aRNases (L2-3, K-2) being effective virus killers have a more specific mode of action. Obtained results clearly demonstrate that aRNases represent a new class of broad-spectrum virus-inactivating agents. [ABSTRACT FROM AUTHOR]

Ribonuclease 1 (RNase1) is an important digestive enzyme that has been used to study the molecular evolutionary and plant-feeding adaptation of mammals. However, the expression patterns and potential biological function of RNase1 in herbivorous fish is not known. Here, we identified RNase1 from five fish species and illuminated the functional diversification and expression of RNase1 in herbivorous Megalobrama amblycephala. The five identified fish RNase1 genes all have the signature motifs of the RNase A superfamily. No expression of Ma-RNase1 was detected in early developmental stages but a weak expression was detected at 120 and 144 hours post-fertilization (hpf). Ma-RNase1 was only expressed in the liver and heart of one-year-old fish but strongly expressed in the liver, spleen, gut, kidney and testis of two-year-old fish. Moreover, the immunostaining localized RNase1 production to multiple tissues of two-year-old fish. A biological functional analysis of the recombinant protein demonstrated that M. amblycephala RNase1 had a relatively strong ribonuclease activity at its optimal pH 6.1, which is consistent with the pH of its intestinal microenvironment. Collectively, these results clearly show that Ma-RNase1 protein has ribonuclease activity and the expression patterns of Ma-RNase1 are dramatically different in one year and two-year-old fish, suggesting the functional differentiation during fish growing. [ABSTRACT FROM AUTHOR]

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Ribonucleases form a large family of enzymes involved in RNA metabolism and are endowed with a broad range of biological functions. Among the different RNase proteins described in the last decades, those belonging to the Rh/T2/S subfamily show the highest degree of evolutionary conservation, suggesting the occurrence of a key critical ancestral role for this protein family. We have recently defined the human RNASET2 gene as a novel member of a group of oncosuppressors called 'tumor antagonizing genes,' whose activity in the control of cancer growth is carried out mainly in vivo. However, to better define the molecular pathways underlying the oncosuppressive properties of this protein, further structural and functional investigations are necessary, and availability of high-quality recombinant RNASET2 is of paramount importance. Here, we describe a multi-step strategy that allows production of highly pure, catalytically competent recombinant RNASET2 in both wild-type and mutant forms. The recombinant proteins that were produced with our purification strategy will be instrumental to perform a wide range of functional assays aimed at dissecting the molecular mechanisms of RNASET2-mediated tumor suppression. [ABSTRACT FROM AUTHOR]

The present study reports the characterisation of a novel ~12-kDa heterodimeric protein, designated as putrin, from the seeds of Putranjiva roxburghii. The purification of putrin to homogeneity was accomplished using DEAE-sepharose where protein was unbound, CM-sepharose and Cibacron blue 3GA where it was bound and appeared as single peak on a size-exclusion chromatography column. A 15 % sodium dodecyl sulphate polyacrylamide electrophoresis gel, under reducing condition, demonstrated that putrin is made of two polypeptide chains of approximately 4.5 and 7.5 kDa. Circular dichroism studies demonstrated the helical nature and conformational stability of protein at increasing temperatures. Putrin exhibited both RNase and DNase activities and exerted antifungal activity but possessed relatively weak translation-inhibitory activity in cell-free system. The cloning and sequence analysis revealed a 414 bp open reading frame encoding a preproprotein of 137 amino acid residues. The amino acid sequence comparisons and phylogenetic analysis of putrin showed significant homology to 2S seed storage family proteins. The results demonstrated that putrin belongs to 2S albumin family and exhibits a spectrum of biotechnologically exploitable functions. [ABSTRACT FROM AUTHOR]

Mycoplasmas are incapable of de novo synthesis of nucleotides and must therefore secrete nucleases in order to replenish the pool of nucleic acid precursors. The nucleolytic activity of mycoplasmas is an important factor in their pathogenicity. Bacterial ribonucleases (RNases) may produce a broad spectrum of biological effects, including antiviral and antitumor activity. Mycoplasma RNases are therefore of interest. In the present work, the capacity of Acholeplasma laidlawii and Mycoplasma hominis for RNase synthesis and secretion was studied. During the stationary growth phase, these organisms were found to synthesize Mg-dependent RNases, with their highest activity detected outside the cells. Localization of A. laidlawii RNases was determined: almost 90% of the RNase activity was found to be associated with the membrane vesicles. Bioinformational analysis revealed homology between the nucleotide sequences of 14 Bacillus subtilis genes encoding the products with RNase activity and the genes of the mycoplasmas under study. Amino acid sequences of 4 A. laidlawii proteins with ribonuclease activity and the Bsn RNase were also established. [ABSTRACT FROM AUTHOR]

Abstract: Dicer cleaves long double strand RNA (dsRNA) into short ones and initiates RNA interference (RNAi), a sequence-specific post-transcriptional gene silencing process. Moreover, it is also involved in the development of cancers. Herein, we report an electrochemical method to study the ribonuclease activity of Dicer for the first time. The designed dsRNA which is thiolated at one end is firstly immobilized on a gold electrode. Square wave voltammetry is then employed to characterize the ribonuclease activity of Dicer after the enzyme digestion of the dsRNA and the subsequent hybridization between short denatured RNA on the electrode and DNA molecules labeled with electrochemical species. This proposed method shows desirable sensitivity and reproducibility, demonstrating the great potential utility for functional studies of Dicer and RNAi in the future. [Copyright &y& Elsevier]

The investigation of the effect of heavy metal salts on the activity of RNases of soybean seedlings inoculated with soybean rhizobia strains showed changes in specific activity and the spectrum of the multiple forms of the enzyme depending on the concentrations of salts and soybean rhizobia strains. [ABSTRACT FROM AUTHOR]

Abstract: Pathogenesis-related protein-4 (PR-4) family is a group of proteins with a Barwin domain in C-terminus and generally thought to be involved in plant defense responses. However, their detailed roles are poorly understood in defense of apple plant against pathogenic infection. In the present study, a new PR-4 gene (designated as MdPR-4) was identified from Malus domestica, and its roles in defense responses of apple were investigated. The open reading frame of MdPR-4 gene is of 447 bp encoding a protein of 148 amino acids with a Barwin domain in C-terminus and a signal peptide of 26 amino acids in N-terminus. Sequence and structural analysis indicated that MdPR-4 protein belongs to class II of PR-4 family. The high-level expression of MdPR-4 was observed in flowers and leaves as revealed by quantitative real time PCR. The temporal expression analysis demonstrated that MdPR-4 expression could be up-regulated by Botryosphaeria dothidea infection and salicylic acid (SA) or methyl jasmonate (MeJA) treatment, but suppressed by diethyldithiocarbamic acid (DIECA). In vitro assays, recombinant MdPR-4 protein exhibited ribonuclease activity specific for single strand RNA and significant inhibition to hyphal growth of three apple pathogenic fungi B. dothidea, Valsa ceratosperma and Glomerella cingulata. Moreover, the inhibition was reduced by the presence of 5′-ADP. Taken all together, the results indicate that MdPR-4 protein is involved in the defense responses of apple against pathogenic attack by directly inhibiting hyphal growth, and the inhibition is correlated with its ribonuclease activity, where as MdPR-4 expression is regulated by both SA and JA signaling pathway. [Copyright &y& Elsevier]

Wild rice might have previously unidentified genes important for disease resistance and stress tolerance in response to biotic and abiotic stresses. A set of subtractive library was constructed both from leaves of wild rice plants, Oryza grandiglumis (CCDD, 2n=48), treated with fungal elicitor and from wounded leaves. A partial fragment that was homologous to PR10 genes from other plant species was identified via suppression subtractive hybridization and cDNA macroarray. The obtained full-length cDNA sequence (OgPRIO) contains an open reading frame of 480 bp nucleotide, encoding 160 amino acids with a predicted molecular mass of 16.944 kDa and an isoelectric point (pi) of 4.91. The multiple alignment analyses showed the higher sequence homology of OgPRIO with PR10 genes identified in rice plants at amino acid level. The OgPRIO mRNA was not expressed by treatment with wounding, jasmonic acid, and salicylic acid, but markedly expressed in leaves treated with protein phosphatase inhibitors cantharidin and endothall, and yeast extract. In addition, the expression of OgPRIO mRNA was induced within 72 h after treatment with probenazole, one of well-known chemical elicitors, and reached the highest level at 144 h. Heterologous expression of OgPRIO caused growth inhibition and seedling lethality in E. colt and Arabidopsis, respectively. Chemically induced OgPRIO expression with glucocorticoid-mediated transcriptional induction system further reconfirmed its lethality on Arabidopsis seedling. In addition, OgPRlO-cxpressing rice plants, Oryzae sativar were resistant against the infection of rice blast fungus, Kiagnaporthe grisea. These results indicate that OgPRIO is involved in probenazole- and microbe associated molecular patterns-mediated disease resistance responses in plants and is a potential gene for developing disease resistance crop plants. [ABSTRACT FROM AUTHOR]

Abstract: PR4 proteins possess antifungal activity against several pathogenic fungi suggesting a pivotal role in defence reactions against plant pathogen attack. We already showed that wheatwin1, a wheat PR protein of class 4, is endowed with ribonuclease activity. In this study we produced three mutants altering the active site and performed comparative analysis with the native protein also in the presence of the ribonuclease inhibitor 5′-ADP. We characterized the RNA binding site and its interaction with 5′-ADP by 3D modelling and docking studies. Moreover, in vitro antifungal assays have been carried out in order to study the relationship between antifungal and ribonuclease activities. Finally, localization of wheatwin1 in Fusarium culmorum spores was evaluated using fluorescence light microscope. [Copyright &y& Elsevier]

Abstract: Human pancreatic ribonuclease (RNase 1) is expressed in many tissues; has several important enzymatic and biological activities, including efficient cleavage of single-stranded RNA, double-stranded RNA and double-stranded RNA–DNA hybrids, digestion of dietary RNA, regulation of vascular homeostasis, inactivation of the HIV, activation of immature dendritic cells and induction of cytokine production; and furthermore shows potential as an anti-tumor agent. The solution structure and dynamics of uncomplexed, wild-type RNase 1 have been determined by NMR spectroscopy methods to better understand these activities. The family of 20 structures determined on the basis of 6115 unambiguous nuclear Overhauser enhancements is well resolved (pairwise backbone RMSD=1.07 Å) and has the classic RNase A type of tertiary structure. Important structural differences compared with previously determined crystal structures of RNase 1 variants or inhibitor-bound complexes are observed in the conformation of loop regions and side chains implicated in the enzymatic as well as biological activities and binding to the cytoplasmic RNase inhibitor. Multiple side chain conformations observed for key surface residues are proposed to be crucial for membrane binding as well as translocation and efficient RNA hydrolysis. 15N–1H relaxation measurements interpreted with the standard and our extended Lipari–Szabo formalism reveal rigid regions and identify more dynamic loop regions. Some of the most dynamic areas are key for binding to the cytoplasmic RNase inhibitor. This finding and the important differences observed between the structure in solution and that bound to the inhibitor are indications that RNase 1 to inhibitor binding can be better described by the “induced fit” model rather than the rigid “lock-into-key” mechanism. Translational diffusion measurements reveal that RNase 1 is predominantly dimeric above 1 mM concentration; the possible implications of this dimeric state for the remarkable biological properties of RNase 1 are discussed. [Copyright &y& Elsevier]

Abstract: A pathogenesis-related (PR) class 10 protein (designated AmPR-10) was first isolated from the Chinese medicinal material Astragalus mongholicus using a combination of affinity chromatography on Zn-chelate Agarose 4B, ion exchange chromatography on QAE Sephadex A-25 and gel filtration on Sephadex G50. The purified AmPR-10 showed a single band with a molecular mass of 17.2kDa in SDS-PAGE. The molecular mass of intact AmPR-10 was determined to be 32.8kDa by gel filtration. Thus, AmPR-10 is a dimeric protein composed of two identical subunits. AmPR-10 was a glycoprotein detected by periodic acid-Schiff (PAS) staining and its neutral carbohydrate content was 13.7%. The carbohydrate was mainly composed of 73.0% (w/w) arabinose, 15.0% (w/w) glucose and 4.8% (w/w) fructose on the basis of high-performance anion exchange chromatography (HPAEC) analysis. Its N-terminal sequence of 15 amino acid residues was determined as GVISFNEETISTVAP, and showed significant sequence homology to some pathogenesis-related (PR) class 10 proteins. This sequence had 80% identity with the PR-10 protein LlPR10.1C from Lupinus luteus (yellow lupine) followed by 73.3% identity with the PR-10 protein PR10.2 from Medicago sativa (alfalfa), suggesting it is a new member of PR-10 proteins. AmPR-10 exhibited ribonuclease (RNase) activity as do some other PR-10 proteins. The optimal pH and temperature for RNase activity were pH 6.0 and 60°C, respectively. The RNase activity was stable within pH 5.0–11.0. It was stable up to 60°C at pH 6.0. The purification and characterization of AmPR-10 in this investigation furnish additional data to the relatively scanty literature pertaining to Astragali radix proteins. [Copyright &y& Elsevier]

A pathogenesis related protein (AhPR10) is identified from a clone of 6-day old Arachis hypogaea L. (peanut) cDNA library. The clone expressed as a ∼20 kDa protein in E. coli. Nucleotide sequence derived amino acid sequence of the coding region shows its homology with PR10 proteins having Betv1 domain and P loop motif. Recombinant AhPR10 has ribonuclease activity, and antifungal activity against the peanut pathogens Fusarium oxysporum and Rhizoctonia solani. Mutant protein AhPR10-K54N where lys54 is mutated to asn54 loses its ribonuclease and antifungal activities. FITC labeled AhPR10 and AhPR10-K54N are internalized by hyphae of F. oxysporum and R. solani but the later protein does not inhibit the fungal growth. This suggests that the ribonuclease function of AhPR10 is essential for its antifungal activity. Energy and temperature dependent internalization of AhPR10 into sensitive fungal hyphae indicate that internalization of the protein occurs through active uptake. [ABSTRACT FROM AUTHOR]

We have demonstrated that wheatwin1, a wheat pathogenesis-related protein of class 4 (PR4), has ribonuclease activity. Both native and recombinant proteins hydrolyse RNA from wheat coleoptils and have antifungal activity. Sepharose-bound wheatwin1 is able to interact with either wheat or Fusarium culmorum RNA. 3D modelling studies showed that, like ribonucleases A and T1, the action mechanism should involve two His residues, an Arg residue and an Asp residue. [Copyright &y& Elsevier]

Many developmental and physiological changes, including alterations of enzyme activities, occur in plants under low temperature stress. In this study the total ribonuclease activity was determined in crude extracts from root tips of soybean seedlings germinated at 25 °C, subjected to chilling conditions (10°C) and recovered at optimal temperature (25°C). Measurements of RNase activity were performed every 24 hours starting from the third to the 10-th day of growth. We found that chilling caused a considerable increase in ribonuclease activity (in comparison with the control), with an activity peak on the fourth day of the cold treatment. The enzyme activity in root extracts of the plants recovered after cold stress decreased along with the time of recovery. No differences were found in approximate molecular weight (35 kDa) and pH optimum (6.0) for ribonucleases extracted from control and chilled soybean roots. [ABSTRACT FROM AUTHOR]

Rhizopus oryzae IFO 4697 was found to produce intracellular ribonuclease (RNase), and its growth and activity could be regulated under selected metal ion stress. The addition of Fe2+, Mg2+ and Zn2+ to the SLSR medium was essential to growth and RNase production. Ca2+ and Mo6+ stimulated RNase production. It is concluded that the addition of 100 mg/ml Ca2+, 5 mg/ml Mo6+, 0.7 mg/ml Zn2+, 2 mg/ml Fe2+, and 49 mg/ml Mg2+ to the SLSR medium was the best condition for producing RNase in high specific activity (3780 U/mg protein). This result indicates that a metal ion-regulated liquid medium is an efficient culture method for RNase production. [ABSTRACT FROM AUTHOR]

An abundant 17 kDa protein which was isolated and characterized from 10-day old healthy root tissue of white lupin ( Lupinus albus) proved to have a high sequence similarity to pathogenesis-related proteins found in other species. Subsequently, a corresponding clone ( LaPR-10) was identified in a cDNA library prepared from the same tissue that exhibited a high amino acid sequence similarity to a number of the PR-10 family proteins. The clone contains an open reading frame encoding a polypeptide of 158 amino acids, with a predicted molecular mass of 16 905 Da and an isoelectric point of 4.66. Southern blot analysis indicates that LaPR-10 is likely a single-copy gene, or a member of a small gene family. The clone was expressed in Escherichia coli, and its protein product was purified to near homogeneity. Both the native and the recombinant proteins were immunorecognized by antibodies raised against pea PR-10 proteins, and exhibited a ribonucleolytic activity against several RNA preparations, including lupin root total RNA. Characterization of its enzymatic properties indicates that the LaPR-10 protein belongs to the class II ribonucleases. We present evidence that the white lupin 17 kDa protein is constitutively expressed during all stages of root development and, to a lesser extent, in other plant parts. In addition, we demonstrate the presence, in the LaPR-10 amino acid sequence, of a number of motifs that are common to most PR-10 proteins, as well as a RGD motif that is shared only with the alfalfa SRG1 sequence. [ABSTRACT FROM AUTHOR]

Ribonuclease assays have revealed, in contrast to the self-incompatibility (SI) system of Nicotiana alata, there is no detectable ribonuclease activity that correlates with the presence of the functional stigmatic S-gene product in Papaver rhoeas. Thus, we have shown that the inhibition of incompatible pollen tube growth in P. rhoeas is not associated with ribonuclease activity. Furthermore, the finding that pollen from P. rhocas, unlike that from N. alata, is insensitive to purified bovine pancreatic ribonuclease A at very high concentrations, suggests that the involvement of ribonucleases in the inhibition reaction of the SI response in P. rhoeas is unlikely. In addition, the level of ribonuclease activity in mature stigmas of P. rhoes is very much tower than that in N. alata and significantly, the level of ribonuclease activity did not rise in conjunction with the developmental expression of SI. Therefore, as a result of these studies, we believe that SI in P. rhoeas does not involve ribonuclease activity. [ABSTRACT FROM AUTHOR]

The amount of protein synthesis in germinating castor-bean seeds has been estimated by the quantitative and qualitative exmainatin of polysomes from the seeds in the presence and absence of gibberellic acid (GA). Careful optimisation of polysome extraction procedures was required to minimise the ribonuclease activity in the extracts. Ribonuclease activity in seed extracts increased fourfold over the first 5 d of germination. Gibberellic acid stimulated polysome formation about twofold during the first 4 d of germination. It also stimulated the amount of mRNA associated with polysomes by about twofold during the first 3 d of germination. Between days 1 and 5 of germination, polysome formation was primarily limited by mRNA availability. During the period 0-24 h, polysome formation was independent of mRNA levles. The increase in enzyme activities stimulated by GA was probably the result of an increase in the amount of cellular mRNA. No evidence was obtained for an action of GA on translation other than on the increased production of RNA. Examination of the recruitment of isocitrate-lyase mRNA into polysomes showed that GA did not specifically stimulate production of this enzyme. [ABSTRACT FROM AUTHOR]

We isolated S allele-associated cDNA clones from each of the stylar cDNA libraries of Lycopersicon peruvianum of two different S genotypes ( SSand S S) with S Sallele-associated cDNA (LPS11) as a probe. The longest cDNA clones, designated LPS12 and LPS13, which were 779 bp and 853 bp in length, contained open reading frames of 189 and 210 amino acids, respectively. The three S alleleassociated cDNAs (LPS11, LPS12, and LPS13) did not cross-hybridize to each other under highly stringent condition by northern blot analysis. Their average identity to Nicotiana alata S-proteins so far was 49%. The fragments corresponding to LPS11 or LPS12 cosegregated with their respective S alleles in genetic crosses. From these results, we conclude that the three cloned cDNAs were derived from the three different S alleles of L. peruvianum. [ABSTRACT FROM AUTHOR]