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15 pages, 2119 KiB

Open AccessArticle

Rapid Discovery of Antimicrobial and Antimalarial Agents from Natural Product Fragments

by Jianying Han, Xueting Liu, Lixin Zhang, Wesley C. Van Voorhis, Ronald J. Quinn and Miaomiao Liu

Separations 2024, 11(7), 194; https://doi.org/10.3390/separations11070194 - 23 Jun 2024

Cited by 1 | Viewed by 1452

Abstract

Fragment-based drug discovery (FBDD) focuses on small compounds, known as fragments, typically with a molecular weight of less than 300 Da. This study highlights the benefits of employing a pure natural product library for FBDD, contrasting with the predominant use of synthetic libraries. [...] Read more.

Fragment-based drug discovery (FBDD) focuses on small compounds, known as fragments, typically with a molecular weight of less than 300 Da. This study highlights the benefits of employing a pure natural product library for FBDD, contrasting with the predominant use of synthetic libraries. Practical methods for rapidly constructing such libraries from crude extracts were demonstrated across various plant and microbial samples. Twenty-nine (29) natural product fragments, including a new compound (20), were identified. Antimicrobial activities were assessed for a subset of the isolated compounds, revealing potent fragments (MICs 4–8 μg/mL) against Mycobacterium bovis bacille Calmette-Guérin (BCG), Staphylococcus aureus (SA), and methicillin-resistant S. aureus (MRSA). Furthermore, a native mass spectrometry technique was introduced to rapidly identify non-competitive fragments against malarial proteins. As a result, two pairs of non-competitive fragments, lepiotin C (31) and 7-amino deacetoxy cephalosporanic acid (32) binding to dynein light chain 1, methyl gallate (33) and β-santanin (34) binding to dUTPase, were identified, serving as promising starting points for developing potent malarial protein inhibitors. Full article

(This article belongs to the Section Analysis of Natural Products and Pharmaceuticals)

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13 pages, 3186 KiB

Open AccessArticle

Bacteriophage T5 dUTPase: Combination of Common Enzymatic and Novel Functions

by Anatoly Glukhov, Victor Marchenkov, Ulyana Dzhus, Antonina Krutilina, Georgii Selikhanov and Azat Gabdulkhakov

Int. J. Mol. Sci. 2024, 25(2), 892; https://doi.org/10.3390/ijms25020892 - 10 Jan 2024

Cited by 2 | Viewed by 1863

Abstract

The main function of dUTPases is to regulate the cellular levels of dUTP and dTTP, thereby playing a crucial role in DNA repair mechanisms. Despite the fact that mutant organisms with obliterated dUTPase enzymatic activity remain viable, it is not possible to completely [...] Read more.

The main function of dUTPases is to regulate the cellular levels of dUTP and dTTP, thereby playing a crucial role in DNA repair mechanisms. Despite the fact that mutant organisms with obliterated dUTPase enzymatic activity remain viable, it is not possible to completely knock out the dut gene due to the lethal consequences of such a mutation for the organism. As a result, it is considered that this class of enzymes performs an additional function that is essential for the organism’s survival. In this study, we provide evidence that the dUTPase of bacteriophage T5 fulfills a supplemental function, in addition to its canonical role. We determined the crystal structure of bacteriophage T5 dUTPase with a resolution of 2.0 Å, and we discovered a distinct short loop consisting of six amino acid residues, representing a unique structural feature specific to the T5-like phages dUTPases. The removal of this element did not affect the overall structure of the homotrimer, but it had significant effects on the development of the phage. Furthermore, it was shown that the enzymatic function and the novel function of the bacteriophage T5 dUTPase are unrelated and independent from each other. Full article

(This article belongs to the Section Molecular Biology)

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11 pages, 4127 KiB

Open AccessArticle

Beta-Hydroxyisovaleryl-Shikonin Eradicates Epithelial Cell Adhesion Molecule-Positive Liver Cancer Stem Cells by Suppressing dUTP Pyrophosphatase Expression

by Yoshiro Asahina, Hajime Takatori, Kouki Nio, Hikari Okada, Takehiro Hayashi, Tomoyuki Hayashi, Tomomi Hashiba, Tsuyoshi Suda, Masaki Nishitani, Saiho Sugimoto, Masao Honda, Shuichi Kaneko and Taro Yamashita

Int. J. Mol. Sci. 2023, 24(22), 16283; https://doi.org/10.3390/ijms242216283 - 14 Nov 2023

Cited by 2 | Viewed by 1740

Abstract

Cancer stem cells (CSCs) play an essential role in tumorigenesis, chemoresistance, and metastasis. Previously, we demonstrated that the development of hepatocellular carcinoma (HCC) is dictated by a subset of epithelial cell adhesion molecule-positive (EpCAM+) liver CSCs with the activation of Wnt signaling. In [...] Read more.

Cancer stem cells (CSCs) play an essential role in tumorigenesis, chemoresistance, and metastasis. Previously, we demonstrated that the development of hepatocellular carcinoma (HCC) is dictated by a subset of epithelial cell adhesion molecule-positive (EpCAM+) liver CSCs with the activation of Wnt signaling. In this study, we evaluated the expression of dUTP pyrophosphatase (dUTPase), which plays a central role in the development of chemoresistance to 5-fluorouracil, in EpCAM+ HCC cells. We further evaluated the effect of beta-hydroxyisovaleryl-shikonin (β-HIVS), an ATP-noncompetitive inhibitor of protein tyrosine kinases, on HCC CSCs. EpCAM and dUTPase were expressed in hepatoblasts in human fetal liver, hepatic progenitors in adult cirrhotic liver, and a subset of HCC cells. Sorted EpCAM+ CSCs from HCC cell lines showed abundant nuclear accumulation of dUTPase compared with EpCAM-negative cells. Furthermore, treatment with the Wnt signaling activator BIO increased EpCAM and dUTPase expression. In contrast, β-HIVS treatment decreased dUTPase expression. β-HIVS treatment decreased the population of EpCAM+ liver CSCs in a dose-dependent manner in vitro and suppressed tumor growth in vivo compared with the control vehicle. Taken together, our data suggest that dUTPase could be a good target to eradicate liver CSCs resistant to 5-fluorouracil. β-HIVS is a small molecule that could decrease dUTPase expression and target EpCAM+ liver CSCs. Full article

(This article belongs to the Special Issue Molecular Mechanisms of Human Liver Diseases 2.0)

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14 pages, 2962 KiB

Open AccessArticle

Molecular Characterization of UL50 (dUTPase) Gene of Bovine Herpes Virus 1

by Farzana Shahin, Sohail Raza, Xi Chen, Changmin Hu, Yingyu Chen, Huanchun Chen and Aizhen Guo

Animals 2023, 13(16), 2607; https://doi.org/10.3390/ani13162607 - 12 Aug 2023

Cited by 2 | Viewed by 2043

Abstract

Bovine herpes virus -1 (BoHV-1) infection leads to upper respiratory tract infection, conjunctivitis and genital disorders in cattle. To control BoHV-1, it is important to understand the role of viral proteins in viral infection. BoHV-1 has several gene products to help in viral [...] Read more.

Bovine herpes virus -1 (BoHV-1) infection leads to upper respiratory tract infection, conjunctivitis and genital disorders in cattle. To control BoHV-1, it is important to understand the role of viral proteins in viral infection. BoHV-1 has several gene products to help in viral replication in infected cell. One such gene is deoxyuridine triphosphate nucleotidohydrolase (dUTPase) also known as UL50. In this study, we analyzed the amino acid sequence of UL50 (dUTPase) using bioinformatics tools and found that it was highly conserved among herpesvirus family. Then, it was cloned and expressed in Escherichia coli Rosetta (DE3), induced by isopropy1-b-D-thiogalactopyranoside (IPTG) and the recombinant UL50 protein was purified to immunize rabbits for the preparation of polyclonal antiserum. The results indicated that the UL50 gene of BoHV-1 was composed of 978 nucleotides, which encoded 323 amino acids. Western blot analysis revealed that polyclonal sera against UL50 reacted with a band of 34 kDa. Furthermore, immunofluorescence assay showed that UL50 localized in the cytoplasmic area. Taken together, UL50 was successfully cloned, expressed and detected in BoHV-1-infected cells and was localized in the cytoplasm to help in the replication of BoHV-1 in infected cells. Full article

(This article belongs to the Special Issue Animal Herpesvirus Infections)

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18 pages, 1787 KiB

Open AccessReview

EBV dUTPase: A Novel Modulator of Inflammation and the Tumor Microenvironment in EBV-Associated Malignancies

by Marshall V. Williams, Irene Mena-Palomo, Brandon Cox and Maria Eugenia Ariza

Cancers 2023, 15(3), 855; https://doi.org/10.3390/cancers15030855 - 30 Jan 2023

Cited by 6 | Viewed by 4044

Abstract

There is increasing evidence that put into question the classical dogma that the Epstein–Barr virus (EBV) exists in cells as either a lytic virus in which new progeny is produced or in a latent state in which no progeny is produced. Notably, a [...] Read more.

There is increasing evidence that put into question the classical dogma that the Epstein–Barr virus (EBV) exists in cells as either a lytic virus in which new progeny is produced or in a latent state in which no progeny is produced. Notably, a third state has now been described, known as the abortive-lytic phase, which is characterized by the expression of some immediate early (IE) and early (E) genes, but no new virus progeny is produced. While the function of these IE and E gene products is not well understood, several recent studies support the concept they may contribute to tumor promotion by altering the tumor microenvironment (TME). The mechanisms by which these viral gene products may contribute to tumorigenesis remain unclear; however, it has been proposed that some of them promote cellular growth, immune evasion, and/or inhibit apoptosis. One of these EBV early gene products is the deoxyuridine triphosphate nucleotidohydrolase (dUTPase) encoded by BLLF3, which not only contributes to the establishment of latency through the production of activin A and IL-21, but it may also alter the TME, thus promoting oncogenesis. Full article

(This article belongs to the Special Issue Study on Tumor Microenvironment in Lymphoma)

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10 pages, 2633 KiB

Open AccessArticle

Deletion of the ASFV dUTPase Gene E165R from the Genome of Highly Virulent African Swine Fever Virus Georgia 2010 Does Not Affect Virus Replication or Virulence in Domestic Pigs

by Elizabeth A. Vuono, Elizabeth Ramirez-Medina, Sarah Pruitt, Ayushi Rai, Nallely Espinoza, Ediane Silva, Lauro Velazquez-Salinas, Douglas P. Gladue and Manuel V. Borca

Viruses 2022, 14(7), 1409; https://doi.org/10.3390/v14071409 - 28 Jun 2022

Cited by 17 | Viewed by 2961

Abstract

African swine fever (ASF) is a frequently lethal disease of domestic and wild swine currently producing a pandemic affecting pig production in Eurasia. The causative agent, ASF virus (ASFV) is a structurally complex virus with a large genome harboring over 150 genes. One [...] Read more.

African swine fever (ASF) is a frequently lethal disease of domestic and wild swine currently producing a pandemic affecting pig production in Eurasia. The causative agent, ASF virus (ASFV) is a structurally complex virus with a large genome harboring over 150 genes. One of them, E165R, encodes for a protein belonging to the dUTPase family. The fine structure of the purified protein has been recently analyzed and its dUTPase activity tested. In addition, it has been reported that a BA71 mutant virus, adapted to growth in Vero cells, lacking the E165R gene presented a drastic decreased replication in swine macrophages, its natural target cell. Herein, we report the development of a recombinant virus, ASFV-G-∆E165R, harboring the deletion of the E165R gene from the genome of the highly virulent field isolate ASFV Georgia 2010 (ASFV-G). Interestingly, ASFV-G-∆E165R replicates in primary swine macrophage cultures as efficiently as the parental virus ASFV-G. In addition, ASFV-G-∆E165R also replicates in experimentally inoculated domestic pigs with equal efficacy as ASFV-G and produced a lethal disease almost indistinguishable from that induced by the parental virus. Therefore, results presented here clearly demonstrated that E165R gene is not essential or important for ASFV replication in swine macrophages nor disease production in domestic pigs. Full article

(This article belongs to the Special Issue Endemic and Emerging Swine Viruses 2022)

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24 pages, 969 KiB

Open AccessReview

Viral dUTPases: Modulators of Innate Immunity

by Maria Eugenia Ariza, Brandon Cox, Britney Martinez, Irene Mena-Palomo, Gloria Jeronimo Zarate and Marshall Vance Williams

Biomolecules 2022, 12(2), 227; https://doi.org/10.3390/biom12020227 - 28 Jan 2022

Cited by 7 | Viewed by 4789

Abstract

Most free-living organisms encode for a deoxyuridine triphosphate nucleotidohydrolase (dUTPase; EC 3.6.1.23). dUTPases represent a family of metalloenzymes that catalyze the hydrolysis of dUTP to dUMP and pyrophosphate, preventing dUTP from being incorporated into DNA by DNA polymerases, maintaining a low dUTP/dTTP pool [...] Read more.

Most free-living organisms encode for a deoxyuridine triphosphate nucleotidohydrolase (dUTPase; EC 3.6.1.23). dUTPases represent a family of metalloenzymes that catalyze the hydrolysis of dUTP to dUMP and pyrophosphate, preventing dUTP from being incorporated into DNA by DNA polymerases, maintaining a low dUTP/dTTP pool ratio and providing a necessary precursor for dTTP biosynthesis. Thus, dUTPases are involved in maintaining genomic integrity by preventing the uracilation of DNA. Many DNA-containing viruses, which infect mammals also encode for a dUTPase. This review will summarize studies demonstrating that, in addition to their classical enzymatic activity, some dUTPases possess novel functions that modulate the host innate immune response. Full article

(This article belongs to the Section Biomacromolecules: Nucleic Acids)

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9 pages, 1156 KiB

Open AccessArticle

Detection and Characterisation of an Endogenous Betaretrovirus in Australian Wild Deer

by Jose L. Huaman, Carlo Pacioni, David M. Forsyth, Anthony Pople, Jordan O. Hampton, Teresa G. Carvalho and Karla J. Helbig

Viruses 2022, 14(2), 252; https://doi.org/10.3390/v14020252 - 27 Jan 2022

Cited by 1 | Viewed by 2896

Abstract

Endogenous retroviruses (ERVs) are the remnants of past retroviral infections that once invaded the host’s germline and were vertically transmitted. ERV sequences have been reported in mammals, but their distribution and diversity in cervids are unclear. Using next-generation sequencing, we identified a nearly [...] Read more.

Endogenous retroviruses (ERVs) are the remnants of past retroviral infections that once invaded the host’s germline and were vertically transmitted. ERV sequences have been reported in mammals, but their distribution and diversity in cervids are unclear. Using next-generation sequencing, we identified a nearly complete genome of an endogenous betaretrovirus in fallow deer (Dama dama). Further genomic analysis showed that this provirus, tentatively named cervid endogenous betaretrovirus 1 (CERV β1), has typical betaretroviral genome features (gag-pro-pol-env) and the betaretrovirus-specific dUTPase domain. In addition, CERV β1 pol sequences were detected by PCR in the six non-native deer species with wild populations in Australia. Phylogenetic analyses demonstrated that CERV β1 sequences from subfamily Cervinae clustered as sister taxa to ERV-like sequences in species of subfamily Muntiacinae. These findings, therefore, suggest that CERV β1 endogenisation occurred after the split of these two subfamilies (between 3.3 and 5 million years ago). Our results provide important insights into the evolution of betaretroviruses in cervids. Full article

(This article belongs to the Section Animal Viruses)

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16 pages, 5136 KiB

Open AccessArticle

Identification and Characterization of a Novel Epitope of ASFV-Encoded dUTPase by Monoclonal Antibodies

by Shuai Zhang, Rui Wang, Xiaojing Zhu, Jiaxin Jin, Wenlong Lu, Xuyang Zhao, Bo Wan, Yifei Liao, Qin Zhao, Christopher L. Netherton, Guoqing Zhuang, Aijun Sun and Gaiping Zhang

Viruses 2021, 13(11), 2175; https://doi.org/10.3390/v13112175 - 28 Oct 2021

Cited by 10 | Viewed by 3556

Abstract

Deoxyuridine 5′-triphosphate nucleotidohydrolase (dUTPase) of African swine fever virus (ASFV) is an essential enzyme required for efficient virus replication. Previous crystallography data have indicated that dUTPase (E165R) may serve as a therapeutic target for inhibiting ASFV replication; however, the specificity of the targeting [...] Read more.

Deoxyuridine 5′-triphosphate nucleotidohydrolase (dUTPase) of African swine fever virus (ASFV) is an essential enzyme required for efficient virus replication. Previous crystallography data have indicated that dUTPase (E165R) may serve as a therapeutic target for inhibiting ASFV replication; however, the specificity of the targeting site(s) in ASFV dUTPase remains unclear. In this study, 19 mouse monoclonal antibodies (mAbs) were produced, in which four mAbs showed inhibitory reactivity against E165R recombinant protein. Epitope mapping studies indicated that E165R has three major antigenic regions: 100–120 aa, 120–140 aa, and 140–165 aa. Three mAbs inhibited the dUTPase activity of E165R by binding to the highly conserved 149–RGEGRFGSTG–158 amino acid sequence. Interestingly, 8F6 mAb specifically recognized ASFV dUTPase but not Sus scrofa dUTPase, which may be due to structural differences in the amino acids of F151, R153, and F154 in the motif V region. In summary, we developed anti-E165R-specific mAbs, and identified an important antibody-binding antigenic epitope in the motif V of ASFV dUTPase. Our study provides a comprehensive analysis of mAbs that target the antigenic epitope of ASFV dUTPase, which may contribute to the development of novel antibody-based ASFV therapeutics. Full article

(This article belongs to the Special Issue Pathogenesis of Swine Fever Virus)

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11 pages, 19039 KiB

Open AccessReview

Structures and Functional Diversities of ASFV Proteins

by Guoguo Wang, Mengjia Xie, Wei Wu and Zhongzhou Chen

Viruses 2021, 13(11), 2124; https://doi.org/10.3390/v13112124 - 21 Oct 2021

Cited by 87 | Viewed by 10389

Abstract

African swine fever virus (ASFV), the causative pathogen of the recent ASF epidemic, is a highly contagious double-stranded DNA virus. Its genome is in the range of 170~193 kbp and encodes 68 structural proteins and over 100 non-structural proteins. Its high pathogenicity strains [...] Read more.

African swine fever virus (ASFV), the causative pathogen of the recent ASF epidemic, is a highly contagious double-stranded DNA virus. Its genome is in the range of 170~193 kbp and encodes 68 structural proteins and over 100 non-structural proteins. Its high pathogenicity strains cause nearly 100% mortality in swine. Consisting of four layers of protein shells and an inner genome, its structure is obviously more complicated than many other viruses, and its multi-layered structures play different kinds of roles in ASFV replication and survival. Each layer possesses many proteins, but very few of the proteins have been investigated at a structural level. Here, we concluded all the ASFV proteins whose structures were unveiled, and explained their functions from the view of structures. Those structures include ASFV AP endonuclease, dUTPases (E165R), pS273R protease, core shell proteins p15 and p35, non-structural proteins pA151R, pNP868R (RNA guanylyltransferase), major capsid protein p72 (gene B646L), Bcl-2-like protein A179L, histone-like protein pA104R, sulfhydryl oxidase pB119L, polymerase X and ligase. These novel structural features, diverse functions, and complex molecular mechanisms promote ASFV to escape the host immune system easily and make this large virus difficult to control. Full article

(This article belongs to the Special Issue Enteric and Respiratory Viruses in Animals)

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17 pages, 1276 KiB

Open AccessFeature PaperReview

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome: The Human Herpesviruses Are Back!

by Maria Eugenia Ariza

Biomolecules 2021, 11(2), 185; https://doi.org/10.3390/biom11020185 - 29 Jan 2021

Cited by 39 | Viewed by 14518

Abstract

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) or Systemic Exertion Intolerance Disease (SEID) is a chronic multisystem illness of unconfirmed etiology. There are currently no biomarkers and/or signatures available to assist in the diagnosis of the syndrome and while numerous mechanisms have been hypothesized to [...] Read more.

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) or Systemic Exertion Intolerance Disease (SEID) is a chronic multisystem illness of unconfirmed etiology. There are currently no biomarkers and/or signatures available to assist in the diagnosis of the syndrome and while numerous mechanisms have been hypothesized to explain the pathology of ME/CFS, the triggers and/or drivers remain unknown. Initial studies suggested a potential role of the human herpesviruses especially Epstein-Barr virus (EBV) in the disease process but inconsistent and conflicting data led to the erroneous suggestion that these viruses had no role in the syndrome. New studies using more advanced approaches have now demonstrated that specific proteins encoded by EBV could contribute to the immune and neurological abnormalities exhibited by a subgroup of patients with ME/CFS. Elucidating the role of these herpesvirus proteins in ME/CFS may lead to the identification of specific biomarkers and the development of novel therapeutics. Full article

(This article belongs to the Special Issue Epstein-Barr Virus Disease Mechanisms and Stress Responses)

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20 pages, 2265 KiB

Open AccessArticle

The Complete Genome of an Endogenous Nimavirus (Nimav-1_LVa) From the Pacific Whiteleg Shrimp Penaeus (Litopenaeus) Vannamei

by Weidong Bao, Kathy F. J. Tang and Acacia Alcivar-Warren

Genes 2020, 11(1), 94; https://doi.org/10.3390/genes11010094 - 14 Jan 2020

Cited by 17 | Viewed by 4642

Abstract

White spot syndrome virus (WSSV), the lone virus of the genus Whispovirus under the family Nimaviridae, is one of the most devastating viruses affecting the shrimp farming industry. Knowledge about this virus, in particular, its evolution history, has been limited, partly due [...] Read more.

White spot syndrome virus (WSSV), the lone virus of the genus Whispovirus under the family Nimaviridae, is one of the most devastating viruses affecting the shrimp farming industry. Knowledge about this virus, in particular, its evolution history, has been limited, partly due to its large genome and the lack of other closely related free-living viruses for comparative studies. In this study, we reconstructed a full-length endogenous nimavirus consensus genome, Nimav-1_LVa (279,905 bp), in the genome sequence of Penaeus (Litopenaeus) vannamei breed Kehai No. 1 (ASM378908v1). This endogenous virus seemed to insert exclusively into the telomeric pentanucleotide microsatellite (TAACC/GGTTA)_n. It encoded 117 putative genes, with some containing introns, such as g012 (inhibitor of apoptosis, IAP), g046 (crustacean hyperglycemic hormone, CHH), g155 (innexin), g158 (Bax inhibitor 1 like). More than a dozen Nimav-1_LVa genes are involved in the pathogen-host interactions. We hypothesized that g046, g155, g158, and g227 (semaphorin 1A like) were recruited host genes for their roles in immune regulation. Sequence analysis indicated that a total of 43 WSSV genes belonged to the ancestral/core nimavirus gene set, including four genes reported in this study: wsv112 (dUTPase), wsv206, wsv226, and wsv308 (nucleocapsid protein). The availability of the Nimav-1_LVa sequence would help understand the genetic diversity, epidemiology, evolution, and virulence of WSSV. Full article

(This article belongs to the Special Issue Selected Papers from the Mobile Genetic Elements Conference, Woods Hole MA 2019)

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19 pages, 4416 KiB

Open AccessArticle

Beyond Chelation: EDTA Tightly Binds Taq DNA Polymerase, MutT and dUTPase and Directly Inhibits dNTPase Activity

by Anna Lopata, Balázs Jójárt, Éva V. Surányi, Enikő Takács, László Bezúr, Ibolya Leveles, Ábris Á. Bendes, Béla Viskolcz, Beáta G. Vértessy and Judit Tóth

Biomolecules 2019, 9(10), 621; https://doi.org/10.3390/biom9100621 - 17 Oct 2019

Cited by 15 | Viewed by 8916

Abstract

EDTA is commonly used as an efficient chelator of metal ion enzyme cofactors. It is highly soluble, optically inactive and does not interfere with most chemicals used in standard buffers making EDTA a common choice to generate metal-free conditions for biochemical and biophysical [...] Read more.

EDTA is commonly used as an efficient chelator of metal ion enzyme cofactors. It is highly soluble, optically inactive and does not interfere with most chemicals used in standard buffers making EDTA a common choice to generate metal-free conditions for biochemical and biophysical investigations. However, the controversy in the literature on metal-free enzyme activities achieved using EDTA or by other means called our attention to a putative effect of EDTA beyond chelation. Here, we show that EDTA competes for the nucleotide binding site of the nucleotide hydrolase dUTPase by developing an interaction network within the active site similar to that of the substrate. To achieve these findings, we applied kinetics and molecular docking techniques using two different dUTPases. Furthermore, we directly measured the binding of EDTA to dUTPases and to two other dNTPases, the Taq polymerase and MutT using isothermal titration calorimetry. EDTA binding proved to be exothermic and mainly enthalpy driven with a submicromolar dissociation constant considerably lower than that of the enzyme:substrate or the Mg:EDTA complexes. Control proteins, including an ATPase, did not interact with EDTA. Our findings indicate that EDTA may act as a selective inhibitor against dNTP hydrolyzing enzymes and urge the rethinking of the utilization of EDTA in enzymatic experiments. Full article

(This article belongs to the Section Cellular Biochemistry)

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18 pages, 3560 KiB

Open AccessArticle

HDX and Native Mass Spectrometry Reveals the Different Structural Basis for Interaction of the Staphylococcal Pathogenicity Island Repressor Stl with Dimeric and Trimeric Phage dUTPases

by Kinga Nyíri, Matthew J. Harris, Judit Matejka, Olivér Ozohanics, Károly Vékey, Antoni J. Borysik and Beáta G. Vértessy

Biomolecules 2019, 9(9), 488; https://doi.org/10.3390/biom9090488 - 14 Sep 2019

Cited by 9 | Viewed by 3948

Abstract

The dUTPase enzyme family plays an essential role in maintaining the genome integrity and are represented by two distinct classes of proteins; the β-pleated homotrimeric and the all-α homodimeric dUTPases. Representatives of both trimeric and dimeric dUTPases are encoded by Staphylococcus aureus phage [...] Read more.

The dUTPase enzyme family plays an essential role in maintaining the genome integrity and are represented by two distinct classes of proteins; the β-pleated homotrimeric and the all-α homodimeric dUTPases. Representatives of both trimeric and dimeric dUTPases are encoded by Staphylococcus aureus phage genomes and have been shown to interact with the Stl repressor protein of S. aureus pathogenicity island SaPIbov1. In the present work we set out to characterize the interactions between these proteins based on a range of biochemical and biophysical methods and shed light on the binding mechanism of the dimeric φNM1 phage dUTPase and Stl. Using hydrogen deuterium exchange mass spectrometry, we also characterize the protein regions involved in the dUTPase:Stl interactions. Based on these results we provide reasonable explanation for the enzyme inhibitory effect of Stl observed in both types of complexes. Our experiments reveal that Stl employs different peptide segments and stoichiometry for the two different phage dUTPases which allows us to propose a functional plasticity of Stl. The malleable character of Stl serves as a basis for the inhibition of both dimeric and trimeric dUTPases. Full article

(This article belongs to the Section Cellular Biochemistry)

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12 pages, 2313 KiB

Open AccessArticle

A Nymphalid-Infecting Group I Alphabaculovirus Isolated from the Major Passion Fruit Caterpillar Pest Dione juno juno (Lepidoptera: Nymphalidae)

by Bergmann Morais Ribeiro, Ethiane Rozo dos Santos, Luana Beló Trentin, Leonardo Assis da Silva, Fernando Lucas de Melo, Elliot Watanabe Kitajima and Daniel M. P. Ardisson-Araújo

Viruses 2019, 11(7), 602; https://doi.org/10.3390/v11070602 - 3 Jul 2019

Cited by 7 | Viewed by 4232

Abstract

Baculoviruses are capable of infecting a wide diversity of insect pests. In the 1990s, the Dione juno nucleopolyhedrovirus (DijuNPV) was isolated from larvae of the major passionfruit defoliator pest Dione juno juno (Nymphalidae) and described at ultrastructural and pathological levels. In this study, [...] Read more.

Baculoviruses are capable of infecting a wide diversity of insect pests. In the 1990s, the Dione juno nucleopolyhedrovirus (DijuNPV) was isolated from larvae of the major passionfruit defoliator pest Dione juno juno (Nymphalidae) and described at ultrastructural and pathological levels. In this study, the complete genome sequence of DijuNPV was determined and analyzed. The circular genome presents 122,075 bp with a G + C content of 50.9%. DijuNPV is the first alphabaculovirus completely sequenced that was isolated from a nymphalid host and may represent a divergent species. It appeared closely related to Orgyia pseudotsugata multiple nucleopolyhedrovirus (OpMNPV) and other Choristoneura-isolated group I alphabaculoviruses. We annotated 153 open reading frames (ORFs), including a set of 38 core genes, 26 ORFs identified as present in lepidopteran baculoviruses, 17 ORFs unique in baculovirus, and several auxiliary genes (e.g., bro, cathepsin, chitinase, iap-1, iap-2, and thymidylate kinase). The thymidylate kinase (tmk) gene was present fused to a dUTPase (dut) gene in other baculovirus genomes. DijuNPV likely lost the dut portion together with the iap-3 homolog. Overall, the genome sequencing of novel alphabaculoviruses enables a wide understanding of baculovirus evolution. Full article

(This article belongs to the Special Issue Emerging Viruses: Surveillance, Prevention, Evolution and Control)

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