Endonuclease V from the archaeon Thermococcus kodakarensis is an inosine-specific ribonuclease

2021 ◽  
Author(s):  
Miyako Shiraishi ◽  
Michihi Hidaka ◽  
Shigenori Iwai
Keyword(s):  
Dna Repair ◽  
Nucleic Acid ◽  
Potential Role ◽  
Biochemical Properties ◽  
Hyperthermophilic Archaeon ◽  
Thermococcus Kodakarensis ◽  
Substrate Preferences ◽  
Endonuclease V ◽  
Domains Of Life

Abstract Endonuclease V (EndoV) is an inosine-specific endonuclease which is highly conserved in all domains of life: Bacteria, Archaea, and Eukarya; and, therefore, may play an important role in nucleic acid processes. It is currently thought that bacterial EndoVs are involved in DNA repair, while eukaryotic EndoVs are involved in RNA editing based on the differences in substrate preferences. However, the role of EndoV proteins, particularly in the archaeal domain, is still poorly understood. Here, we explored the biochemical properties of EndoV from the hyperthermophilic archaeon Thermococcus kodakarensis (TkoEndoV). We show that TkoEndoV has a strong preference for RNA over DNA. Further, we synthesized 1-methylinosine-containing RNA which is a simple TΨC loop mimic of archaeal tRNA and found that TkoEndoV discriminates between 1-methylinosine and inosine, and selectively acts on inosine. Our findings suggest a potential role of archaeal EndoV in regulation of inosine-containing RNA.

Abstract 2636: Nonmelanoma skin cancer as a marker of a cancer-prone phenotype: Potential role of DNA repair gene variants

2012 ◽  
Author(s):  
Ingo Ruczinski ◽  
Timothy Jorgensen ◽  
Yin Yao Shugart ◽  
Yvette Berthier-Schaad ◽  
Bailey Kessing ◽  
...  
Keyword(s):  
Dna Repair ◽  
Skin Cancer ◽  
Potential Role ◽  
Nonmelanoma Skin Cancer ◽  
Gene Variants ◽  
Repair Gene ◽  
Dna Repair Gene

scholarly journals Resolution of two ADP-ribosylation factor 1 GTPase-activating proteins from rat liver

1997 ◽  
Vol 324 (2) ◽  
pp. 413-419 ◽  
Author(s):  
Paul A. RANDAZZO
Keyword(s):  
Dissociation Constant ◽  
Rat Liver ◽  
Potential Role ◽  
Membrane Traffic ◽  
Adp Ribosylation ◽  
Substrate Preferences ◽  
Gtpase Activating Proteins ◽  
Gtp Binding ◽  
Adp Ribosylation Factor

ADP-ribosylation factor 1 (ARF1) is a 21 kDa GTP-binding protein that regulates multiple steps in membrane traffic. Here, two ARF1 GTPase-activating proteins (GAPs) from rat liver were resolved. The GAPs were antigenically distinct. One reacted with a polyclonal antibody raised against the GAP catalytic peptide previously purified by Makler et al. [Makler, Cukierman, Rotman, Admon and Cassel (1995) J. Biol. Chem. 270, 5232–5237], and here is referred to as GAP1. The other GAP (GAP2) did not react with the antibody. These GAPs differed in phospholipid dependencies. GAP1 was activated 3–7-fold by the acid phospholipids phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidic acid (PA) and phosphatidylserine (PS). In contrast, GAP2 was stimulated 20–40-fold by PIP2. PA and PS had no effect by themselves but PA increased GAP2 activity in the presence of PIP2. The GAPs were otherwise similar in activity. In the presence of phosphoinositides, the Km of GAP1 for ARF1–GTP was estimated to be 8.1±1.6 μM and the dissociation constant for ARF1–guanosine 5′,3-O-(thio)triphosphate (GTP[S]) was 7.4±2.2 μM. GAP2 was similar with a Km for ARF1–GTP of 5.4±1.2 μM and a dissociation constant for ARF1–GTP[S] of 4.8±0.3 μM. Similarly, no differences were found in substrate preferences. Both GAP1 and GAP2 used ARF1 and ARF5 as substrates but not ARF6 or ARF-like protein-2. The potential role of multiple ARF GAPs in the independent regulation of ARF at specific steps in membrane traffic is discussed.

scholarly journals TK1211 Encodes an Amino Acid Racemase towards Leucine and Methionine in the Hyperthermophilic Archaeon Thermococcus kodakarensis

2021 ◽  
Author(s):  
Ren-Chao Zheng ◽  
Xia-Feng Lu ◽  
Hiroya Tomita ◽  
Shin-ichi Hachisuka ◽  
Yu-Guo Zheng ◽  
...  
Keyword(s):  
Amino Acid ◽  
Surface Glycoprotein ◽  
Lag Phase ◽  
Class Iii ◽  
Cell Surface Glycoprotein ◽  
Hyperthermophilic Archaeon ◽  
Thermococcus Kodakarensis ◽  
Domains Of Life ◽  
Amino Acid Racemase

Members of Thermococcales harbor a number of genes encoding putative aminotransferase Class III enzymes. Here, we characterized the TK1211 protein from the hyperthermophilic archaeon Thermococcus kodakarensis. The TK1211 gene was expressed in T. kodakarensis under the control of the strong, constitutive promoter of the cell-surface glycoprotein gene TK0895 (Pcsg). The purified protein did not display aminotransferase activity, but exhibited racemase activity. An examination on most amino acids indicated that the enzyme was a racemase with relatively high activity toward Leu and Met. Kinetic analysis indicated that Leu was the most preferred substrate. A TK1211 gene disruption strain (ΔTK1211) was constructed and grown on minimal media supplemented with l- or d-Leu or l- or d-Met. The wild-type T. kodakarensis is not able to synthesize Leu and displays Leu auxotrophy, providing a direct means to examine the Leu racemase activity of the TK1211 protein in vivo. When we replaced l-Leu with d-Leu in the medium, the host strain with an intact TK1211 gene displayed an extended lag phase, but displayed cell yield similar to that observed in medium with l-Leu. By contrast, the ΔTK1211 strain displayed growth in medium with l-Leu, but could not grow with d-Leu. The results indicate that TK1211 encodes a Leu racemase that is active in T. kodakarensis cells, and that no other protein exhibits this activity, at least to an extent that can support growth. Growth experiments with l- or d-Met also confirmed the Met racemase activity of the TK1211 protein in T. kodakarensis. IMPORTANCE Phylogenetic analysis of aminotransferase Class III proteins from all domains of life reveals numerous groups of protein sequences. One of these groups includes a large number of sequences from Thermococcales species and can be divided into four subgroups. Representatives of three of these subgroups have been characterized in detail. This study reveals that a representative from the remaining uncharacterized subgroup is an amino acid racemase with preference toward Leu and Met. Taken together with results of previous studies on enzymes from Pyrococcus horikoshii and Thermococcus kodakarensis, members of the four subgroups can now be presumed to function as a broad substrate specificity amino acid racemase (Subgroup 1), alanine/serine racemase (Subgroup 2), ornithine ω-aminotransferase (Subgroup 3), or Leu/Met racemase (Subgroup 4).

scholarly journals Identification and enzymatic analysis of an archaeal ATP-dependent serine kinase from the hyperthermophilic archaeon Staphylothermus marinus

2021 ◽  
Author(s):  
Yasunobu Mori ◽  
Hiroki Kawamura ◽  
Takaaki Sato ◽  
Takayuki Fujita ◽  
Ryuhei Nagata ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Sugar Metabolism ◽  
Inorganic Polyphosphates ◽  
Serine Kinase ◽  
Hyperthermophilic Archaeon ◽  
Thermococcus Kodakarensis ◽  
Wide Range ◽  
Genes Encoding ◽  
Phosphate Donor ◽  
Domains Of Life

Serine kinase catalyzes the phosphorylation of free serine (Ser) to produce O -phosphoserine (Sep). An ADP-dependent Ser kinase in the hyperthermophilic archaeon Thermococcus kodakarensis ( Tk -SerK) is involved in cysteine (Cys) biosynthesis and most likely Ser assimilation. An ATP-dependent Ser kinase in the mesophilic bacterium Staphylococcus aureus is involved in siderophore biosynthesis. Although proteins displaying various degrees of similarity with Tk -SerK are distributed in a wide range of organisms, it is unclear if they are actually Ser kinases. Here we examined proteins from Desulfurococcales species in Crenarchaeota that display moderate similarity with Tk -SerK from Euryarchaeota (42-45% identical). Tk - serK homologs from Staphylothermus marinus (Smar_0555), Desulfurococcus amylolyticus (DKAM_0858), and Desulfurococcus mucosus (Desmu_0904) were expressed in Escherichia coli . All three partially purified recombinant proteins exhibited Ser kinase activity utilizing ATP rather than ADP as a phosphate donor. Purified Smar_0555 protein displayed activity towards l -Ser, but not with other compounds including d -Ser, l -threonine and l -homoserine. The enzyme utilized ATP, UTP, GTP, CTP, and the inorganic polyphosphates triphosphate and tetraphosphate as the phosphate donor. Kinetic analysis indicated that the Smar_0555 protein preferred nucleoside 5’-triphosphates compared to triphosphate as a phosphate donor. Transcript levels and Ser kinase activity in S. marinus cells grown with or without serine suggested that the Smar_0555 gene is constitutively expressed. The genes encoding Ser kinases examined here form an operon with genes most likely responsible for the conversion between Sep and 3-phosphoglycerate of central sugar metabolism, suggesting that the ATP-dependent Ser kinases from Desulfurococcales play a role in the assimilation of Ser. IMPORTANCE Homologs of the ADP-dependent Ser kinase from the archaeon Thermococcus kodakarensis ( Tk -SerK) include representatives from all three domains of life. The results of this study show that even homologs from the archaeal order Desulfurococcales, which are the most structurally related to the ADP-dependent Ser kinases from the Thermococcales, are Ser kinases that utilize ATP, and in at least some cases inorganic polyphosphates, as the phosphate donor. The differences in properties between the Desulfurococcales and Thermococcales enzymes raise the possibility that Tk -SerK homologs constitute a group of kinases that phosphorylate free serine with a wide range of phosphate donors.

scholarly journals Potential role of ZEB1 as a DNA repair regulator in colorectal cancer cells revealed by cancer-associated promoter profiling

2017 ◽  
Vol 38 (4) ◽  
pp. 1941-1948 ◽  
Author(s):  
Miao Wang ◽  
Su-Fei He ◽  
Lei-Lei Liu ◽  
Xiao-Xia Sun ◽  
Fan Yang ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Dna Repair ◽  
Cancer Cells ◽  
Potential Role ◽  
Colorectal Cancer Cells

scholarly journals Direct, gabapentin-insensitive interaction of a soluble form of the calcium channel subunit α2δ-1 with thrombospondin-4

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Ehab El-Awaad ◽  
Galyna Pryymachuk ◽  
Cora Fried ◽  
Jan Matthes ◽  
Jörg Isensee ◽  
...  
Keyword(s):  
Neuropathic Pain ◽  
Potential Role ◽  
Binding Assay ◽  
Molecular Level ◽  
Biochemical Properties ◽  
Matrix Proteins ◽  
Soluble Form ◽  
Voltage Gated Calcium Channels ◽  
Thrombospondin 4

Abstract The α2δ‐1 subunit of voltage-gated calcium channels binds to gabapentin and pregabalin, mediating the analgesic action of these drugs against neuropathic pain. Extracellular matrix proteins from the thrombospondin (TSP) family have been identified as ligands of α2δ‐1 in the CNS. This interaction was found to be crucial for excitatory synaptogenesis and neuronal sensitisation which in turn can be inhibited by gabapentin, suggesting a potential role in the pathogenesis of neuropathic pain. Here, we provide information on the biochemical properties of the direct TSP/α2δ-1 interaction using an ELISA-style ligand binding assay. Our data reveal that full-length pentameric TSP-4, but neither TSP-5/COMP of the pentamer-forming subgroup B nor TSP-2 of the trimer-forming subgroup A directly interact with a soluble variant of α2δ-1 (α2δ-1S). Interestingly, this interaction is not inhibited by gabapentin on a molecular level and is not detectable on the surface of HEK293-EBNA cells over-expressing α2δ‐1 protein. These results provide biochemical evidence that supports a specific role of TSP-4 among the TSPs in mediating the binding to neuronal α2δ‐1 and suggest that gabapentin does not directly target TSP/α2δ-1 interaction to alleviate neuropathic pain.

Structure of human endonuclease V as an inosine-specific ribonuclease

2014 ◽  
Vol 70 (9) ◽  
pp. 2286-2294 ◽  
Author(s):  
Zhemin Zhang ◽  
Zhitai Hao ◽  
Zhong Wang ◽  
Qing Li ◽  
Wei Xie
Keyword(s):  
Metal Binding ◽  
Binding Pocket ◽  
Rnase H ◽  
Structural Basis ◽  
Phosphodiester Bond ◽  
Substrate Preferences ◽  
Cysteine Motif ◽  
Endonuclease V ◽  
Cleavage Efficiency ◽  
Domains Of Life

The 6-aminopurine ring of adenosine (A) can be deaminated to form the 6-oxopurine of inosine (I). Endonuclease Vs (EndoVs) are inosine-specific nucleases that cleave at the second phosphodiester bond 3′ to inosine. EndoV proteins are highly conserved in all domains of life, but the bacterial and human enzymes seem to display distinct substrate preferences. While the bacterial enzymes exhibit high cleavage efficiency on various nucleic acid substrates, human EndoV (hEndoV) is most active towards ssRNA but is much less active towards other substrates. However, the structural basis of substrate recognition by hEndoV is not well understood. In this study, the 2.3 Å resolution crystal structure of hEndoV was determined and its unusual RNA-cleaving properties were investigated. The enzyme preserves the general `RNase H-like' structure, especially in the wedge motif, the metal-binding site and the hypoxanthine-binding pocket. hEndoV also features several extra insertions and a characteristic four-cysteine motif, in which Cys227 and Cys228, two cysteines that are highly conserved in higher eukaryotes, play important roles in catalysis. The structure presented here helps in understanding the substrate preference of hEndoV catalysis.

scholarly journals miRNAs in gastrointestinal diseases: can we effectively deliver RNA-based therapeutics orally?

Nanomedicine ◽  
2019 ◽  
Vol 14 (21) ◽  
pp. 2873-2889 ◽  
Author(s):  
A K M Nawshad Hossian ◽  
Gerardo G Mackenzie ◽  
George Mattheolabakis
Keyword(s):  
Nucleic Acid ◽  
Oral Delivery ◽  
Potential Role ◽  
Gastrointestinal Diseases ◽  
The Novel ◽  
Development And Utilization ◽  
Cancer And Inflammation ◽  
Carrier Systems

Nucleic acid-based therapeutics are evaluated for their potential of treating a plethora of diseases, including cancer and inflammation. Short nucleic acids, such as miRNAs, have emerged as versatile regulators for gene expression and are studied for therapeutic purposes. However, their inherent instability in vivo following enteral and parenteral administration has prompted the development of novel methodologies for their delivery. Although research on the oral delivery of siRNAs is progressing, with the development and utilization of promising carrier-based methodologies for the treatment of a plethora of gastrointestinal diseases, research on miRNA-based oral therapeutics is lagging behind. In this review, we present the potential role of miRNAs in diseases of the GI tract, and analyze current research and the cardinal features of the novel carrier systems used for nucleic acid oral delivery that can be expanded for oral miRNA administration.

Potential role of dna repair genes in lung cancer development in chromium exposed individuals

2014 ◽  
Vol 229 ◽  
pp. S157
Author(s):  
Erika Halasova ◽  
Tatiana Matakova ◽  
Maria Skerenova ◽  
Mirko Halasa ◽  
Silvia Javorkova ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Dna Repair ◽  
Potential Role ◽  
Cancer Development ◽  
Dna Repair Genes ◽  
Repair Genes

scholarly journals The Role of Nucleases and Nucleic Acid Editing Enzymes in the Regulation of Self-Nucleic Acid Sensing

2021 ◽  
Vol 12 ◽  
Author(s):  
Pauline Santa ◽  
Anne Garreau ◽  
Lee Serpas ◽  
Amandine Ferriere ◽  
Patrick Blanco ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Expression Profiles ◽  
Genetic Material ◽  
Biochemical Properties ◽  
Therapeutic Strategies ◽  
Innate Immune ◽  
Novel Therapeutic Strategies ◽  
Turn Over

Detection of microbial nucleic acids by the innate immune system is mediated by numerous intracellular nucleic acids sensors. Upon the detection of nucleic acids these sensors induce the production of inflammatory cytokines, and thus play a crucial role in the activation of anti-microbial immunity. In addition to microbial genetic material, nucleic acid sensors can also recognize self-nucleic acids exposed extracellularly during turn-over of cells, inefficient efferocytosis, or intracellularly upon mislocalization. Safeguard mechanisms have evolved to dispose of such self-nucleic acids to impede the development of autoinflammatory and autoimmune responses. These safeguard mechanisms involve nucleases that are either specific to DNA (DNases) or RNA (RNases) as well as nucleic acid editing enzymes, whose biochemical properties, expression profiles, functions and mechanisms of action will be detailed in this review. Fully elucidating the role of these enzymes in degrading and/or processing of self-nucleic acids to thwart their immunostimulatory potential is of utmost importance to develop novel therapeutic strategies for patients affected by inflammatory and autoimmune diseases.

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