scholarly journals Initiation of cytosolic plant purine nucleotide catabolism involves a monospecific xanthosine monophosphate phosphatase

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Katharina J. Heinemann ◽  
Sun-Young Yang ◽  
Henryk Straube ◽  
Nieves Medina-Escobar ◽  
Marina Varbanova-Herde ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Arabidopsis Thaliana ◽  
Substrate Specificity ◽  
Vascular Plants ◽  
Mutational Analysis ◽  
Adenosine Monophosphate ◽  
Guanosine Monophosphate ◽  
Purine Nucleotide ◽  
High Substrate

AbstractIn plants, guanosine monophosphate (GMP) is synthesized from adenosine monophosphate via inosine monophosphate and xanthosine monophosphate (XMP) in the cytosol. It has been shown recently that the catabolic route for adenylate-derived nucleotides bifurcates at XMP from this biosynthetic route. Dephosphorylation of XMP and GMP by as yet unknown phosphatases can initiate cytosolic purine nucleotide catabolism. Here we show that Arabidopsis thaliana possesses a highly XMP-specific phosphatase (XMPP) which is conserved in vascular plants. We demonstrate that XMPP catalyzes the irreversible entry reaction of adenylate-derived nucleotides into purine nucleotide catabolism in vivo, whereas the guanylates enter catabolism via an unidentified GMP phosphatase and guanosine deaminase which are important to maintain purine nucleotide homeostasis. We also present a crystal structure and mutational analysis of XMPP providing a rationale for its exceptionally high substrate specificity, which is likely required for the efficient catalysis of the very small XMP pool in vivo.

159 EFFECT OF 3-ISOBUTYL-1-METHYLXANTHINE (IBMX) ON THE GERMINAL VESICLE STAGE OF PORCINE OOCYTES DURING OOCYTE COLLECTION IN VITRO

2014 ◽  
Vol 26 (1) ◽  
pp. 193
Author(s):  
R. Appeltant ◽  
J. Beek ◽  
D. Maes ◽  
A. Van Soom
Keyword(s):  
Germinal Vesicle ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Cumulus Cells ◽  
Guanosine Monophosphate ◽  
Developmental Competence ◽  
Meiotic Arrest ◽  
Oocyte Collection

When using modern maturation conditions for in vitro maturation, pig oocytes yield ~20% blastocysts only. One problem is that cumulus cells, which are normally connected with the immature oocyte by cellular projections penetrating through the zona pellucida and with the oolemma via gap junctions, are prematurely losing these connections after the cumulus–oocyte complex is removed from the follicle. The oocyte possesses a type 3 phosphodiesterase, which degrades 3′,5′-cyclic adenosine monophosphate (cAMP), and this activity is inhibited by supply of 3′,5′-cyclic guanosine monophosphate (cGMP) to the oocyte via the cumulus cells. Consequently, cAMP levels, which are typically high during early stages of oocyte maturation in vivo, decrease, leading to spontaneous nuclear maturation and oocytes of low developmental competence. Therefore, the maintenance of these cumulus-oocyte connections is important to keep cAMP high and the oocyte under meiotic arrest. One way to prevent this drop in cAMP is using N6, 2′-o-dibutyryladenosine 3′,5′-cyclic monophosphate sodium (dbcAMP) that causes an arrest at germinal vesicle (GV) stage II (Funahashi et al. 1997 Biol. Reprod. 57, 49–53). Another option is collecting the oocytes in a medium containing the phoshodiesterase inhibitor, IBMX. The present study investigated the influence of IBMX on the progression of the GV of the oocyte after collection, just before the start of the maturation procedure. The GV stage was defined according to Sun et al. (2004 Mol. Reprod. Dev. 69, 228–234). In parallel with the findings on dbcAMP, we hypothesised an arrest at GV II by the presence of IBMX during collection. One group of oocytes were collected in HEPES-buffered TALP without IBMX (n = 375) and another group in the same medium containing 0.5 mM IBMX (n = 586). An average incubation time of 140 min was applied in both groups, and 3 replicates were performed. The proportions of oocytes before or at GV II and beyond GV II were compared in both groups using logistic regression analysis. The proportion of oocytes was included as dependent variable and group (IBMX addition or not) as independent variable. Replicate was also included in the model. The proportion of oocytes before or at GV II was not statistically significant between the group without and the group with IBMX (59.2 v. 58.7% respectively; P > 0.05). In conclusion, the use of IBMX during oocyte collection did not influence the state of the germinal vesicle of the oocyte during collection, indicating that IBMX did not cause a meiotic arrest in the oocytes during collecting in vitro.

scholarly journals cGAS-mediated autophagy protects the liver from ischemia-reperfusion injury independently of STING

2018 ◽  
Vol 314 (6) ◽  
pp. G655-G667 ◽  
Author(s):  
Zhao Lei ◽  
Meihong Deng ◽  
Zhongjie Yi ◽  
Qian Sun ◽  
Richard A. Shapiro ◽  
...  
Keyword(s):  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Adenosine Monophosphate ◽  
Cyclic Guanosine Monophosphate ◽  
Protective Role ◽  
Guanosine Monophosphate ◽  
Independent Manner

Liver ischemia-reperfusion (I/R) injury occurs through induction of oxidative stress and release of damage-associated molecular patterns (DAMPs), including cytosolic DNA released from dysfunctional mitochondria or from the nucleus. Cyclic guanosine monophosphate–adenosine monophosphate (cGAMP) synthase (cGAS) is a cytosolic DNA sensor known to trigger stimulator of interferon genes (STING) and downstream type 1 interferon (IFN-I) pathways, which are pivotal innate immune system responses to pathogen. However, little is known about the role of cGAS/STING in liver I/R injury. We subjected C57BL/6 (WT), cGAS knockout (cGAS−/−), and STING-deficient (STINGgt/gt) mice to warm liver I/R injury and that found cGAS−/− mice had significantly increased liver injury compared with WT or STINGgt/gt mice, suggesting a protective effect of cGAS independent of STING. Liver I/R upregulated cGAS in vivo and also in vitro in hepatocytes subjected to anoxia/reoxygenation (A/R). We confirmed a previously published finding that hepatocytes do not express STING under normoxic conditions or after A/R. Hepatocytes and liver from cGAS−/− mice had increased cell death and reduced induction of autophagy under hypoxic conditions as well as increased apoptosis. Protection could be restored in cGAS−/− hepatocytes by overexpression of cGAS or by pretreatment of mice with autophagy inducer rapamycin. Our findings indicate a novel protective role for cGAS in the regulation of autophagy during liver I/R injury that occurs independently of STING. NEW & NOTEWORTHY Our studies are the first to document the important role of cGAS in the acute setting of sterile injury induced by I/R. Specifically, we provide evidence that cGAS protects liver from I/R injury in a STING-independent manner.

scholarly journals Crystal Structures of [Fe]-Hydrogenase from Methanolacinia paynteri Suggest a Path of the FeGP-Cofactor Incorporation Process

Inorganics ◽  
2020 ◽  
Vol 8 (9) ◽  
pp. 50
Author(s):  
Gangfeng Huang ◽  
Francisco Javier Arriaza-Gallardo ◽  
Tristan Wagner ◽  
Seigo Shima
Keyword(s):  
Crystal Structure ◽  
Protein Structure ◽  
Guanosine Monophosphate ◽  
Amino Group ◽  
Prosthetic Group ◽  
Closed Conformation ◽  
Open Conformation ◽  
Local Protein Structure ◽  
Local Protein

[Fe]-hydrogenase (Hmd) catalyzes the reversible heterolytic cleavage of H2, and hydride transfer to methenyl-tetrahydromethanopterin (methenyl-H4MPT+). The iron-guanylylpyridinol (FeGP) cofactor, the prosthetic group of Hmd, can be extracted from the holoenzyme and inserted back into the protein. Here, we report the crystal structure of an asymmetric homodimer of Hmd from Methanolacinia paynteri (pHmd), which was composed of one monomer in the open conformation with the FeGP cofactor (holo-form) and a second monomer in the closed conformation without the cofactor (apo-form). In addition, we report the symmetric pHmd-homodimer structure in complex with guanosine monophosphate (GMP) or guanylylpyridinol (GP), in which each ligand was bound to the protein, where the GMP moiety of the FeGP-cofactor is bound in the holo-form. Binding of GMP and GP modified the local protein structure but did not induce the open conformation. The amino-group of the Lys150 appears to interact with the 2-hydroxy group of pyridinol ring in the pHmd–GP complex, which is not the case in the structure of the pHmd–FeGP complex. Lys150Ala mutation decreased the reconstitution rate of the active enzyme with the FeGP cofactor at the physiological pH. These results suggest that Lys150 might be involved in the FeGP-cofactor incorporation into the Hmd protein in vivo.

scholarly journals Crystal structure of Arabidopsis thaliana casein kinase 2 α1

2020 ◽  
Vol 76 (4) ◽  
pp. 182-191
Author(s):  
Manon Demulder ◽  
Lieven De Veylder ◽  
Remy Loris
Keyword(s):  
Crystal Structure ◽  
Arabidopsis Thaliana ◽  
Model System ◽  
Casein Kinase ◽  
Casein Kinase 2 ◽  
Body Motion ◽  
Biological Functions ◽  
Α Subunit ◽  
Crystal Forms

Casein kinase 2 (CK2) is a ubiquitous pleiotropic enzyme that is highly conserved across eukaryotic kingdoms. CK2 is singular amongst kinases as it is highly rigid and constitutively active. Arabidopsis thaliana is widely used as a model system in molecular plant research; the biological functions of A. thaliana CK2 are well studied in vivo and many of its substrates have been identified. Here, crystal structures of the α subunit of A. thaliana CK2 in three crystal forms and of its complex with the nonhydrolyzable ATP analog AMppNHp are presented. While the C-lobe of the enzyme is highly rigid, structural plasticity is observed for the N-lobe. Small but significant displacements within the active cleft are necessary in order to avoid steric clashes with the AMppNHp molecule. Binding of AMppNHp is influenced by a rigid-body motion of the N-lobe that was not previously recognized in maize CK2.

scholarly journals Structural basis for the substrate specificity and catalytic features of pseudouridine kinase from Arabidopsis thaliana

2020 ◽  
Author(s):  
Sang-Hoon Kim ◽  
Claus-Peter Witte ◽  
Sangkee Rhee
Keyword(s):  
Arabidopsis Thaliana ◽  
Substrate Specificity ◽  
Conformational Transition ◽  
Functional Divergence ◽  
Rna Modification ◽  
Structural Basis ◽  
Translation Efficiency ◽  
Active Site Residues ◽  
Dynamic Features

Abstract RNA modifications can regulate the stability of RNAs, mRNA–protein interactions, and translation efficiency. Pseudouridine is a prevalent RNA modification, and its metabolic fate after RNA turnover was recently characterized in eukaryotes, in the plant Arabidopsis thaliana. Here, we present structural and biochemical analyses of PSEUDOURIDINE KINASE from Arabidopsis (AtPUKI), the enzyme catalyzing the first step in pseudouridine degradation. AtPUKI, a member of the PfkB family of carbohydrate kinases, is a homodimeric α/β protein with a protruding small β-strand domain, which serves simultaneously as dimerization interface and dynamic substrate specificity determinant. AtPUKI has a unique nucleoside binding site specifying the binding of pseudourine, in particular at the nucleobase, by multiple hydrophilic interactions, of which one is mediated by a loop from the small β-strand domain of the adjacent monomer. Conformational transition of the dimerized small β-strand domains containing active site residues is required for substrate specificity. These dynamic features explain the higher catalytic efficiency for pseudouridine over uridine. Both substrates bind well (similar Km), but only pseudouridine is turned over efficiently. Our studies provide an example for structural and functional divergence in the PfkB family and highlight how AtPUKI avoids futile uridine phosphorylation which in vivo would disturb pyrimidine homeostasis.

scholarly journals Pulmonary surfactant–biomimetic nanoparticles potentiate heterosubtypic influenza immunity

Science ◽  
2020 ◽  
Vol 367 (6480) ◽  
pp. eaau0810 ◽  
Author(s):  
Ji Wang ◽  
Peiyu Li ◽  
Yang Yu ◽  
Yuhong Fu ◽  
Hongye Jiang ◽  
...  
Keyword(s):  
Pulmonary Surfactant ◽  
Adenosine Monophosphate ◽  
Cyclic Guanosine Monophosphate ◽  
Alveolar Epithelial Cells ◽  
Guanosine Monophosphate ◽  
H1n1 Vaccine ◽  
Alveolar Epithelial ◽  
Heterosubtypic Immunity ◽  
Biomimetic Nanoparticles

Current influenza vaccines only confer protection against homologous viruses. We synthesized pulmonary surfactant (PS)–biomimetic liposomes encapsulating 2′,3′-cyclic guanosine monophosphate–adenosine monophosphate (cGAMP), an agonist of the interferon gene inducer STING (stimulator of interferon genes). The adjuvant (PS-GAMP) vigorously augmented influenza vaccine–induced humoral and CD8+ T cell immune responses in mice by simulating the early phase of viral infection without concomitant excess inflammation. Two days after intranasal immunization with PS-GAMP–adjuvanted H1N1 vaccine, strong cross-protection was elicited against distant H1N1 and heterosubtypic H3N2, H5N1, and H7N9 viruses for at least 6 months while maintaining lung-resident memory CD8+ T cells. Adjuvanticity was then validated in ferrets. When alveolar epithelial cells (AECs) lacked Sting or gap junctions were blocked, PS-GAMP–mediated adjuvanticity was substantially abrogated in vivo. Thus, AECs play a pivotal role in configuring heterosubtypic immunity.

Structural and biochemical characterization of the catalytic domains of GdpP reveals a unified hydrolysis mechanism for the DHH/DHHA1 phosphodiesterase

2018 ◽  
Vol 475 (1) ◽  
pp. 191-205 ◽  
Author(s):  
Feng Wang ◽  
Qing He ◽  
Kaixuan Su ◽  
Tiandi Wei ◽  
Sujuan Xu ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Catalytic Mechanism ◽  
Biochemical Characterization ◽  
Adenosine Monophosphate ◽  
Guanosine Monophosphate ◽  
Catalytic Domains ◽  
Membrane Bound ◽  
Nucleotide Specificity ◽  
Binding Position

The Asp-His-His and Asp-His-His-associated (DHH/DHHA1) domain-containing phosphodiesterases (PDEs) that catalyze degradation of cyclic di-adenosine monophosphate (c-di-AMP) could be subdivided into two subfamilies based on the final product [5′-phosphadenylyl-adenosine (5′-pApA) or AMP]. In a previous study, we revealed that Rv2837c, a stand-alone DHH/DHHA1 PDE, employs a 5′-pApA internal flipping mechanism to produce AMPs. However, why the membrane-bound DHH/DHHA1 PDE can only degrade c-di-AMP to 5′-pApA remains obscure. Here, we report the crystal structure of the DHH/DHHA1 domain of GdpP (GdpP-C), and structures in complex with c-di-AMP, cyclic di-guanosine monophosphate (c-di-GMP), and 5′-pApA. Structural analysis reveals that GdpP-C binds nucleotide substrates quite differently from how Rv2837c does in terms of substrate-binding position. Accordingly, the nucleotide-binding site of the DHH/DHHA1 PDEs is organized into three (C, G, and R) subsites. For GdpP-C, in the C and G sites c-di-AMP binds and degrades into 5′-pApA, and its G site determines nucleotide specificity. To further degrade into AMPs, 5′-pApA must slide into the C and R sites for flipping and hydrolysis as in Rv2837c. Subsequent mutagenesis and enzymatic studies of GdpP-C and Rv2837c uncover the complete flipping process and reveal a unified catalytic mechanism for members of both DHH/DHHA1 PDE subfamilies.

scholarly journals Challenges on Cyclic Nucleotide Phosphodiesterases Imaging with Positron Emission Tomography: Novel Radioligands and (Pre-)Clinical Insights since 2016

2021 ◽  
Vol 22 (8) ◽  
pp. 3832
Author(s):  
Susann Schröder ◽  
Matthias Scheunemann ◽  
Barbara Wenzel ◽  
Peter Brust
Keyword(s):  
Positron Emission Tomography ◽  
Cyclic Nucleotide ◽  
Intracellular Signaling ◽  
Adenosine Monophosphate ◽  
Guanosine Monophosphate ◽  
Emission Tomography ◽  
Cyclic Nucleotide Phosphodiesterases ◽  
Positron Emission ◽  
Nucleotide Phosphodiesterases

Cyclic nucleotide phosphodiesterases (PDEs) represent one of the key targets in the research field of intracellular signaling related to the second messenger molecules cyclic adenosine monophosphate (cAMP) and/or cyclic guanosine monophosphate (cGMP). Hence, non-invasive imaging of this enzyme class by positron emission tomography (PET) using appropriate isoform-selective PDE radioligands is gaining importance. This methodology enables the in vivo diagnosis and staging of numerous diseases associated with altered PDE density or activity in the periphery and the central nervous system as well as the translational evaluation of novel PDE inhibitors as therapeutics. In this follow-up review, we summarize the efforts in the development of novel PDE radioligands and highlight (pre-)clinical insights from PET studies using already known PDE radioligands since 2016.

Polydatin Attenuates Carbachol-induced Contraction of Guinea Pig Gallbladder

2019 ◽  
Vol 18 (1) ◽  
pp. 34-38
Author(s):  
Chen Lei ◽  
Pan Xiang ◽  
Shen Yonggang ◽  
Song Kai ◽  
Zhong Xingguo ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Guinea Pig ◽  
Smooth Muscles ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Cyclic Guanosine Monophosphate ◽  
Guanosine Monophosphate ◽  
Dependent Manner ◽  
Underlying Mechanisms ◽  
Dose Dependent

The aim of this study was to determine whether polydatin, a glucoside of resveratrol isolated from the root of Polygonum cuspidatum, warranted development as a potential therapeutic for ameliorating the pain originating from gallbladder spasm disorders and the underlying mechanisms. Guinea pig gallbladder smooth muscles were treated with polydatin and specific inhibitors to explore the mechanisms underpinning polydatin-induced relaxation of carbachol-precontracted guinea pig gallbladder. Our results shown that polydatin relaxed carbachol-induced contraction in a dose-dependent manner through the nitric oxide/cyclic guanosine monophosphate/protein kinase G and the cyclic adenosine monophosphate/protein kinase A signaling pathways as well as the myosin light chain kinase and potassium channels. Our findings suggested that there was value in further exploring the potential therapeutic use of polydatin in gallbladder spasm disorders.

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