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Proceedings of the National Academy of... Jun 1976Crystals of solid phase-derived semisynthetic ribonuclease-S' were prepared and compared with those for native ribonuclease-S' and -S. The semisynthetic species used was...
Crystals of solid phase-derived semisynthetic ribonuclease-S' were prepared and compared with those for native ribonuclease-S' and -S. The semisynthetic species used was the noncovalent complex of synthetic fragment-(1-20), corresponding to residues 1 through 20 of bovine pancreatic ribonuclease-A (ribonucleate 3'-pyrimidino-oligonucleotidohydrolase, EC 3.1.4.22), and native ribonuclease-S-(21-124); the fragment containing residues 21 through 124 of ribonuclease-A. This semisynthetic complex was completely active enzymatically, was homogeneous as judged by polyacrylamide gel electrophoresis, and had no greater than trace amounts of excess ribonuclease-s(21-124) as judged by affinity chromatography. Crystallization of both semisynthetic and native ribonuclease-s' at pH 5.3 resulted in well-formed crystallseater than trace amounts of excess ribonuclease-S-T21-124) as judged by affinity chromatography. Crystallization of both semisynthetic and native ribonuclease-S' at pH 5.3 resulted in well-formed crystals with the symmetry of space group P3121 and unit cell dimensions a=b=44.82, c=97.3 A. This crystal form corresponds to the Y form of native ribonuclease-S previously reported [Wyckoff et al. (1967) J. Biol. Chem. 242, 3749-3753]. X-ray diffraction patterns of the crystals were indistinguishable, indicative of the structural identity of semisynthetic and native ribonuclease-S'.
Topics: Crystallography; Protein Conformation; Ribonucleases; X-Ray Diffraction
PubMed: 1064856
DOI: 10.1073/pnas.73.6.1844 -
Biochemistry Sep 2020Glycosylation is a common modification that can endow proteins with altered physical and biological properties. Ribonuclease 1 (RNase 1), which is the human homologue of...
Glycosylation is a common modification that can endow proteins with altered physical and biological properties. Ribonuclease 1 (RNase 1), which is the human homologue of the archetypal enzyme RNase A, undergoes N-linked glycosylation at asparagine residues 34, 76, and 88. We have produced the three individual glycoforms that display the core heptasaccharide, ManGlcNAc, and analyzed the structure of each glycoform by using small-angle X-ray scattering along with molecular dynamics simulations. The glycan on Asn34 is relatively compact and rigid, donates hydrogen bonds that "cap" the carbonyl groups at the C-terminus of an α-helix, and enhances protein thermostability. In contrast, the glycan on Asn88 is flexible and can even enter the enzymic active site, hindering catalysis. The N-glycosylation of Asn76 has less pronounced consequences. These data highlight the diverse behaviors of ManGlcNAc pendants and provide a structural underpinning to the functional consequences of protein glycosylation.
Topics: Catalytic Domain; Glycosylation; Humans; Models, Molecular; Nitrogen; Ribonucleases
PubMed: 32544330
DOI: 10.1021/acs.biochem.0c00191 -
Molecules (Basel, Switzerland) Nov 2021Barnase is an extracellular ribonuclease secreted by that was originally studied as a small stable enzyme with robust folding. The identification of barnase... (Review)
Review
Barnase is an extracellular ribonuclease secreted by that was originally studied as a small stable enzyme with robust folding. The identification of barnase intracellular inhibitor barstar led to the discovery of an incredibly strong protein-protein interaction. Together, barnase and barstar provide a fully genetically encoded toxin-antitoxin pair having an extremely low dissociation constant. Moreover, compared to other dimerization systems, the barnase-barstar module provides the exact one-to-one ratio of the complex components and possesses high stability of each component in a complex and high solubility in aqueous solutions without self-aggregation. The unique properties of barnase and barstar allow the application of this pair for the engineering of different variants of targeted anticancer compounds and cytotoxic supramolecular complexes. Using barnase in suicide gene therapy has also found its niche in anticancer therapy. The application of barnase and barstar in contemporary experimental cancer therapy is reflected in the review.
Topics: Bacillus; Bacterial Proteins; Drug Delivery Systems; Humans; Kinetics; Models, Molecular; Nanotechnology; Neoplasms; Protein Conformation; Ribonucleases
PubMed: 34833876
DOI: 10.3390/molecules26226785 -
Biomolecules Dec 2020Bacterial ribonuclease binase exhibits a cytotoxic effect on tumor cells possessing certain oncogenes. The aim of this study was to identify the structural parts of the...
Bacterial ribonuclease binase exhibits a cytotoxic effect on tumor cells possessing certain oncogenes. The aim of this study was to identify the structural parts of the binase molecule that exert cytotoxicity. Out of five designed peptides, the peptides representing the binase regions 21-50 and 74-94 have the highest cytotoxic potential toward human cervical HeLa and breast BT-20 and MCF-7 cancer cells. The peptides B21-50 and B74-94 were not able to enter human lung adenocarcinoma A549 cells, unlike BT-20 cells, explaining their failure to inhibit A549 cell proliferation. The peptide B74-94 shares similarities with epidermal growth factor (EGF), suggesting the peptide's specificity for EGF receptor overexpressed in BT-20 cells. Thus, the binase-derived peptides have the potential of being further developed as tumor-targeting peptides.
Topics: Apoptosis; Cell Proliferation; Endoribonucleases; HeLa Cells; Humans; MCF-7 Cells; Neoplasms; Peptides; Ribonucleases
PubMed: 33375305
DOI: 10.3390/biom11010016 -
Nature Communications May 2022RNase H2 is a specialized enzyme that degrades RNA in RNA/DNA hybrids and deficiency of this enzyme causes a severe neuroinflammatory disease, Aicardi Goutières...
RNase H2 is a specialized enzyme that degrades RNA in RNA/DNA hybrids and deficiency of this enzyme causes a severe neuroinflammatory disease, Aicardi Goutières syndrome (AGS). However, the molecular mechanism underlying AGS is still unclear. Here, we show that RNase H2 is associated with a subset of genes, in a transcription-dependent manner where it interacts with RNA Polymerase II. RNase H2 depletion impairs transcription leading to accumulation of R-loops, structures that comprise RNA/DNA hybrids and a displaced DNA strand, mainly associated with short and intronless genes. Importantly, accumulated R-loops are processed by XPG and XPF endonucleases which leads to DNA damage and activation of the immune response, features associated with AGS. Consequently, we uncover a key role for RNase H2 in the transcription of human genes by maintaining R-loop homeostasis. Our results provide insight into the mechanistic contribution of R-loops to AGS pathogenesis.
Topics: Autoimmune Diseases of the Nervous System; DNA; DNA Breaks; Endoribonucleases; Humans; Inflammation; Nervous System Malformations; R-Loop Structures; RNA; Ribonuclease H; Ribonuclease, Pancreatic; Ribonucleases
PubMed: 35618715
DOI: 10.1038/s41467-022-30604-0 -
The FEBS Journal Oct 2011RNases are enzymes that cleave RNAs, resulting in remarkably diverse biological consequences. Many RNases are cytotoxic. In some cases, they attack selectively malignant... (Review)
Review
RNases are enzymes that cleave RNAs, resulting in remarkably diverse biological consequences. Many RNases are cytotoxic. In some cases, they attack selectively malignant cells triggering an apoptotic response. A number of eukaryotic and bacterial RNase-based strategies are being developed for use in anticancer and antiviral therapy. However, the physiological functions of these RNases are often poorly understood. This review focuses on the properties of the extracellular RNases from Bacillus amyloliquefaciens (barnase) and Bacillus intermedius (binase), the characteristics of their biosynthesis regulation and their physiological role, with an emphasis on the similarities and differences. Barnase and binase can be regarded as molecular twins according to their highly similar structure, physical-chemical and catalytic properties. Nevertheless, the 'life paths' of these enzymes are not the same, as their expression in bacteria is controlled by diverse signals. Binase is predominantly synthesized under phosphate starvation, whereas barnase production is strictly dependent on the multifunctional Spo0A regulator controlling sporulation, biofilm formation and cannibalism. Barnase and binase also have some distinctions in practical applications. Barnase was initially suggested to be useful in research and biotechnology as a tool for studying protein-protein interactions, for RNA elimination from biological samples, for affinity purification of RNase fusion proteins, for the development of cloning vectors and for sterility acquisition by transgenic plants. Binase, as later barnase, was tested for antiviral, antitumour and immunogenic effects. Both RNases have found their own niche in cancer research as a result of success in targeted delivery and selectivity towards tumour cells.
Topics: Amino Acid Sequence; Animals; Bacillus; Bacterial Proteins; Base Sequence; Endoribonucleases; Humans; Models, Molecular; Molecular Sequence Data; Protein Structure, Tertiary; RNA; Ribonucleases; Sequence Alignment
PubMed: 21824291
DOI: 10.1111/j.1742-4658.2011.08294.x -
Current Pharmaceutical Biotechnology Jun 2008Onconase (ONC) is an amphibian member of the bovine pancreatic ribonuclease (RNase A) superfamily that exhibits innate antitumoral activity. ONC has been granted both... (Review)
Review
Onconase (ONC) is an amphibian member of the bovine pancreatic ribonuclease (RNase A) superfamily that exhibits innate antitumoral activity. ONC has been granted both orphan-drug and fast-track status by the U.S. Food and Drug Administration for the treatment of malignant mesothelioma, and is poised to become the first chemotherapeutic agent based on a ribonuclease. Investigations into the mechanism of ribonuclease-based cytotoxicity have elucidated several important determinants for cytotoxicity, including efficient deliverance of ribonucleolytic activity to the cytosol and preservation of conformation stability. Nevertheless, the most striking similarity between ONC and bovine seminal ribonuclease, another naturally cytotoxic ribonuclease, is their insensitivity to inhibition by the potent cytosolic ribonuclease inhibitor protein (RI). RI typically binds to its ribonuclease ligands with femtomolar affinity--an extraordinary feat considering the modest sequence identity among the bound ribonucleases. Mammalian ribonucleases such as RNase A or its human homologue, RNase 1, have the potential to be more attractive chemotherapeutic agents than ONC owing to their higher catalytic activity, low potential for immunogenicity, favorable tissue distribution, and high therapeutic index, but are limited by their sensitivity to RI. These non-toxic mammalian ribonucleases can be transformed into potent cytotoxins by engendering them with RI-evasion using protein engineering strategies such as site-directed mutagenesis, multimerization, fusion to a targeting moiety, and chemical modification. In several instances, these engineered ribonucleases exhibit greater cytotoxicity in vitro than does ONC. Herein, we review the biochemical characteristics of RIribonuclease complexes and progress towards the development of mammalian ribonuclease-based chemotherapeutics through the elicitation of RI-evasion.
Topics: Amino Acid Sequence; Animals; Antineoplastic Agents; Cytosol; Enzyme Stability; Humans; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Protein Binding; Ribonucleases; Sequence Alignment
PubMed: 18673284
DOI: 10.2174/138920108784567344 -
Protein Expression and Purification Feb 2022Combinations of ribonucleases (RNases) are commonly used to digest RNA into oligoribonucleotide fragments prior to liquid chromatography-mass spectrometry (LC-MS)...
Combinations of ribonucleases (RNases) are commonly used to digest RNA into oligoribonucleotide fragments prior to liquid chromatography-mass spectrometry (LC-MS) analysis. The distribution of the RNase target sequences or nucleobase sites within an RNA molecule is critical for achieving a high mapping coverage. Cusativin and MC1 are nucleotide-specific endoribonucleases encoded in the cucumber and bitter melon genomes, respectively. Their high specificity for cytidine (Cusativin) and uridine (MC1) make them ideal molecular biology tools for RNA modification mapping. However, heterogenous recombinant expression of either enzyme has been challenging because of their high toxicity to expression hosts and the requirement of posttranslational modifications. Here, we present two highly efficient and time-saving protocols that overcome these hurdles and enhance the expression and purification of these RNases. We first purified MC1 and Cusativin from bacteria by expressing and shuttling both enzymes to the periplasm as MBP-fusion proteins in T7 Express lysY/IE. coli strain at low temperature. The RNases were enriched using amylose affinity chromatography, followed by a subsequent purification via a C-terminal 6xHIS tag. This fast, two-step purification allows for the purification of highly active recombinant RNases significantly surpassing yields reported in previous studies. In addition, we expressed and purified a Cusativin-CBD fusion enzyme in P. pastoris using chitin magnetic beads. Both Cusativin variants exhibited a similar sequence preference, suggesting that neither posttranslational modifications nor the epitope-tags have a substantial effect on the sequence specificity of the enzyme.
Topics: Endoribonucleases; Escherichia coli; Gene Expression; Recombinant Proteins; Ribonucleases
PubMed: 34637916
DOI: 10.1016/j.pep.2021.105987 -
Nature Chemical Biology Apr 2021Cas12g, the type V-G CRISPR-Cas effector, is an RNA-guided ribonuclease that targets single-stranded RNA substrate. The CRISPR-Cas12g system offers a potential platform...
Cas12g, the type V-G CRISPR-Cas effector, is an RNA-guided ribonuclease that targets single-stranded RNA substrate. The CRISPR-Cas12g system offers a potential platform for transcriptome engineering and diagnostic applications. We determined the structures of Cas12g-guide RNA complexes in the absence and presence of target RNA by cryo-EM to a resolution of 3.1 Å and 4.8 Å, respectively. Cas12g adopts a bilobed structure with miniature REC2 and Nuc domains, whereas the guide RNAs fold into a flipped 'F' shape, which is primarily recognized by the REC lobe. Target RNA and the CRISPR RNA (crRNA) guide form a duplex that inserts into the central cavity between the REC and NUC lobes, inducing conformational changes in both lobes to activate Cas12g. The structural insights would facilitate the development of Cas12g-based applications.
Topics: Bacterial Proteins; CRISPR-Associated Proteins; CRISPR-Cas Systems; Clustered Regularly Interspaced Short Palindromic Repeats; Cryoelectron Microscopy; RNA, Bacterial; RNA, Guide, CRISPR-Cas Systems; Ribonucleases
PubMed: 33495647
DOI: 10.1038/s41589-020-00721-2 -
Molekuliarnaia Biologiia 2014Many ribonucleases (RNases) are able to inhibit the reproduction of viruses in infected cell cultures and laboratory animals, but molecular mechanisms of their antiviral... (Review)
Review
Many ribonucleases (RNases) are able to inhibit the reproduction of viruses in infected cell cultures and laboratory animals, but molecular mechanisms of their antiviral activity remain unclear. The review observes the most known RNases which possess established antiviral effects, actually intracellular RNases (RNase L, MCPIPI protein, eosinophylic RNases) as well as exogenously applied ones (RNase A, BS-RNase, onconase, binase, synthetic RNases). Attention is given on two important but not always obligatory aspects in molecule of RNases, which have antiviral properties: catalytic activity and ability to the dimerization. The hypothetic scheme of virus elimination by exogenous RNases, that reflects possible types of interaction of viruses and RNases with a cell, is proposed. The evidence for RNases as classical components of immune defense which are perspective agents for development of new antiviral therapeutics is produced.
Topics: Amphibians; Animals; Antiviral Agents; Bacterial Proteins; Endoribonucleases; Fungal Proteins; Humans; Ribonuclease, Pancreatic; Ribonucleases; Transcription Factors
PubMed: 25842855
DOI: No ID Found