Isolation and partial characterization of the ADP-ribosylated nuclear proteins from Ehrlich ascites tumor cells

The (ADP-ribose)_n protein conjugates formed by incubation of Ehrlich ascites tumor cell nuclei with 1 mM (³H)NAD were isolated by chromatography on boronate cellulose columns with a yield of >85%. Possible contamination by glycoproteins was excluded by rechromatography after specific release of the (ADP-ribose)_n residues from their acceptors. Dodecyl sulfate gel electrophoresis revealed numerous protein bands which coincided with the (³H)ADP-ribose bands obtained by fluorography of the gels. 40% of the acceptor proteins were identified as the nucleosomal core histones. Most of these histones, however, appeared in the non-histone fraction because of extensive modification by poly(ADP-ribose). Drastic changes in properties were also seen in the true non-histone proteins which comprised 60% of the total conjugated protein. Besides several prominent acceptor proteins (M_r = 12,000; 31,000; 125,000) numerous proteins were detected indicating a considerable heterogeneity of non-histone acceptors.     

Rapid determination of chain length pattern in poly(ADP-ribose) samples

(³H)poly(ADP-ribose) synthesized from nuclei by incubation with (³H)NAD was released from protein by alkaline treatment and electrophoresed in dodecyl sulfate gels. Individual polymers up to at least 33 units were completely separated according to their chain length. Size distribution was visualized by fluorography of the gels, and quantified by radioactivity determination of sliced gels The method could be applied to crude nuclear extracts. It showed that nuclei of Ehrlich ascites tumor cells produced a poly(ADP-ribose) pattern distinctly different from that of rat liver nuclei.     

Activation of poly(adenosine diphosphate ribose) polymerase with UV irradiated and UV endonuclease treated SV 40 minichromosome

SV 40 minichromosomes were used as a molecular model of eukaryotic chromatin to probe the nature of the lesion responsible for UV stimulation of poly (ADPR) polymerase. UV irradiation of the minichromosomes with doses between 50 and 1000 J/m² did not increase their ability to stimulate the activity of purified poly(ADPR) polymerase. In contrast, when the minichromosomes were UV irradiated and then treated with $\text{M.}\text{luteus}$ UV endonuclease, there was a marked increase in their ability to stimulate poly(ADPR) polymerase. This stimulation was completely suppressed when histone Hl was added to the poly(ADPR) polymerase assay. These studies demonstrate in a purified $\text{in}\text{vitro}$ system that damage caused by UV irradiation alone is not sufficient to stimulate poly(ADPR) polymerase activity. Only when DNA is nicked at the site of UV damage by UV endonuclease is there stimulation of poly(ADPR) polymerase.     

Synthesis of Simple Adenosine Diphosphate Ribose Analogues

The synthesis of analogues of adenosine diphosphate ribose and acetylated adenosine diphosphate ribose, modified at the northern pentose, is reported. The stereochemistry at the acetylated centers was chosen to minimize acetyl migration and dictated the overall synthetic strategy.     

Roles and mechanisms of the CD38/cyclic adenosine diphosphate ribose/Ca~(2+) signaling pathway

Mobilization of intracellular Ca2+ stores is involved inmany diverse cell functions, including: cell proliferation;differentiation; fertilization; muscle contraction; secre-tion of neurotransmitters, hormones and enzymes;and lymphocyte activation and proliferation. Cyclic ad-enosine diphosphate ribose(cADPR) is an endogenousCa2+ mobilizing nucleotide present in many cell typesand species, from plants to animals. cADPR is formedby ADP-ribosyl cyclases from nicotinamide adenine di-nucleotide. The main ADP-ribosyl cyclase in mammalsis CD38, a multi-functional enzyme and a type Ⅱ mem-brane protein. It has been shown that many extracel-lular stimuli can induce cADPR production that leadsto calcium release or influx, establishing cADPR as asecond messenger. cADPR has been linked to a widevariety of cellular processes, but the molecular mecha-nisms regarding cADPR signaling remain elusive. Theaim of this review is to summarize the CD38/cADPR/Ca2+ signaling pathway, focusing on the recent advanc-es involving the mechanism and physiological functionsof cADPR-mediated Ca2+ mobilization.     

Detecting clinical activity in systemic lupus erythematosus with an archaeal poly(ADP-ribose) polymerase-like thermozyme: a pivotal study

The clinical usefulness of an immunotest was evaluated by using purified poly(adenosine diphosphate (ADP)-ribose) polymerase from Sulfolobus solfataricus (PARPSso) as an antigen to detect the presence of abnormal anti-PARP antibodies in the sera of patients with systemic lupus erythematosus (SLE) at different clinical stages. Sera from 44 patients with SLE, subgrouped on the basis of disease activity (16 with inactive disease, 28 with active disease) were analysed with a new immunotest to detect anti-PARP antibodies, and with an immunofluorescent (IIF) assay for antinuclear antibodies (ANA) detection. ANA detection by IIF revealed that sera of healthy subjects were negative, whereas sera from patients with SLE were positive in all cases (13 positive at 1:80, 15 at 1:160, 15 at 1:320, 1 at 1:640, v/v). Anti-PARP activity was higher in ANA-positive patients than in controls (p?=?0.005). Within the group of SLE sera, disease and anti-PARP activity was increased more significantly in patients with active than in those with inactive disease (p?p?=?0.001, respectively). Correlation between anti-PARP and disease activity in SLE patients was statistically significant (p?Sso seems to be suitable for detecting anti-PARP antibodies and could play a role as a serological marker of disease activity in patients with SLE.     

The crystal structure of type III effector protein XopQ from Xanthomonas oryzae complexed with adenosine diphosphate ribose

Effector proteins are virulence factors that promote pathogenesis by interfering with various cellular events and are delivered directly into host cells by the secretion systems of many Gram‐negative bacteria. Type III effector protein XOO4466 from the plant pathogen Xanthomonas oryzae pv. oryzae (XopQ^Xoo) and XopQ homologs from other phytopathogens have been predicted to be nucleoside hydrolases based on their sequence similarities. However, despite such similarities, recent structural and functional studies have revealed that XopQ^Xoo does not exhibit the expected activity of a nucleoside hydrolase. On the basis of the conservation of a Ca²⁺ coordination shell of a ribose‐binding site and the spacious active site in XopQ^Xoo, we hypothesized that a novel compound containing a ribosyl moiety could serve as a substrate for XopQ^Xoo. Here, we report the crystal structure of XopQ^Xoo in complex with adenosine diphosphate ribose (ADPR), which is involved in regulating cytoplasmic Ca²⁺ concentrations in eukaryotic cells. ADPR is bound to the active site of XopQ^Xoo with its ribosyl end tethered to the Ca²⁺ coordination shell. The binding of ADPR is further stabilized by interactions mediated by hydrophobic residues that undergo ligand‐induced conformational changes. These data showed that XopQ^Xoo is capable of binding a novel chemical bearing a ribosyl moiety, thereby providing the first step toward understanding the functional role of XopQ^Xoo. Proteins 2014; 82:2910–2914. © 2014 Wiley Periodicals, Inc.     

Retinal release from opsin in molecular dynamics simulations

The crystal structures of opsin in the ligand-free and the G-protein-interacting states showed two inter-helical openings between transmembrane (TM) helices TM1 and TM7 and between TM5 and TM6 near the extracellular side that were thought to serve as the retinal uptake and release gates. However, it is unclear which opening is for 11-cis-retinal uptake or all-trans-retinal release although speculations have been proposed based on the structural features of opsin and retinal. In this work, we simulated the exit process of all-trans-retinal from the ligand-free opsin structure by the classical molecular dynamics (MD) and random acceleration molecular dynamics (RAMD). In the 64 ns classical MD simulation, retinal remained in the receptor but moved significantly toward the TM5-TM6 opening and almost inserted into the opening after 50 ns. Complete exit was observed in 114 out of 160 RAMD trajectories with the TM5-TM6 opening being the predominant egress gate while egress from the TM1-TM7 opening was observed in only a few trajectories when relatively large acceleration was applied and large structural alteration of the protein resulted. These results suggest that photolyzed all-trans-retinal is likely released through the TM5-TM6 opening. Based on the unidirectional mechanism of retinal exchange suggested by experiment, we speculate that the TM1-TM7 opening serves as the 11-cis-retinal uptake gate. The spatial occupancy maps of retinal computed from the 160 RAMD trajectories further indicated that retinal experienced significant interactions with the receptor during the exit process. The implications of these findings for disease mechanisms of rhodopsin mutants are discussed.     

Structure of the sirtuin‐linked macrodomain SAV0325 from Staphylococcus aureus

Cells use the post‐translational modification ADP‐ribosylation to control a host of biological activities. In some pathogenic bacteria, an operon‐encoded mono‐ADP‐ribosylation cycle mediates response to host‐induced oxidative stress. In this system, reversible mono ADP‐ribosylation of a lipoylated target protein represses oxidative stress response. An NAD⁺‐dependent sirtuin catalyzes the single ADP‐ribose (ADPr) addition, while a linked macrodomain‐containing protein removes the ADPr. Here we report the crystal structure of the sitruin‐linked macrodomain protein from Staphylococcus aureus, SauMacro (also known as SAV0325) to 1.75‐Å resolution. The monomeric SauMacro bears a previously unidentified Zn²⁺‐binding site that putatively aids in substrate recognition and catalysis. An amino‐terminal three‐helix bundle motif unique to this class of macrodomain proteins provides a structural scaffold for the Zn²⁺ site. Structural features of the enzyme further indicate a cleft proximal to the Zn²⁺ binding site appears well suited for ADPr binding, while a deep hydrophobic channel in the protein core is suitable for binding the lipoate of the lipoylated protein target.     

Adenosine analogs bearing phosphate isosteres as human MDO1 ligands

The human O-acetyl-ADP-ribose deacetylase MDO1 is a mono-ADP-ribosylhydrolase involved in the reversal of post-translational modifications. Until now MDO1 has been poorly characterized, partly since no ligand is known besides adenosine nucleotides. Here, we synthesized thirteen compounds retaining the adenosine moiety and bearing bioisosteric replacements of the phosphate at the ribose 5′-oxygen. These compounds are composed of either a squaryldiamide or an amide group as the bioisosteric replacement and/or as a linker. To these groups a variety of substituents were attached such as phenyl, benzyl, pyridyl, carboxyl, hydroxy and tetrazolyl. Biochemical evaluation showed that two compounds, one from both series, inhibited ADP-ribosyl hydrolysis mediated by MDO1 in high concentrations.     

Elicitor-mediated induction of anthraquinone biosynthesis and regulation of isopentenyl diphosphate isomerase and farnesyl diphosphate synthase activities in cell suspension cultures of Cinchona robusta How.

Treatment of a Cinchona robusta How. cell suspension culture with a homogenate of Phytophthora cinnamomi resulted in cessation of growth and a rapid induction of the biosynthesis of anthraquinone-type phytoalexins. The strongest induction of anthraquinone biosynthesis was obtained when the elicitor was added in the early growth phase of the growth cycle. The accumulation of anthraquinones was accompanied by a tri-phasic response in the activity of isopentenyl diphosphate (IPP) isomerase (EC 5.3.3.2): phase I was characterised by a rapid induction of activity, reaching a maximum at 12 h after elicitation. During phase II, IPP isomerase rapidly decreased to levels below those found in untreated cells. At phase III, IPP isomerase activity increased again, reaching a second maximum at about 72 h after elicitation. During phase I, the activity of farnesyl diphosphate synthase (EC 2.5.1.10) was found to be suppressed. Extraction and assay conditions were optimised for IPP isomerase. The presence of Mn²⁺ in the incubation buffer resulted in a marked increase in the activity of the enzymes obtained from cells in phase I. The induction of IPP isomerase in combination with a concomitant inhibition of farnesyl diphosphate synthase might result in an efficient channeling of C₅-precursors into phytoalexin biosynthesis. Received: 23 August 1996 / Accepted: 20 March 1997     

Catalytic mechanism of type 2 isopentenyl diphosphate:dimethylallyl diphosphate isomerase: verification of a redox role of the flavin cofactor in a reaction with no net redox change

Type 2 isopentenyl diphosphate:dimethylallyl diphosphate isomerase requires redox co-enzymes, i.e., flavin mononucleotide (FMN) and NAD(P)H, for activity, although it catalyzes a non-redox reaction. Spectrometric studies and enzyme assays under anaerobic conditions indicate that FMN is reduced through the reaction and is sufficient for activity. The sole function of NAD(P)H appears to be the reduction of FMN since it could be replaced by an alternate reducing agent. When the enzyme was reconstructed with a flavin analogue, no activity was detected, suggesting that the isomerase reaction proceeds via a radical transfer mechanism.     

Inhibition of monoterpene cyclases by inert analogues of geranyl diphosphate and linalyl diphosphate

The tightly coupled nature of the reaction sequence catalyzed by monoterpene synthases has prevented direct observation of the topologically required isomerization step leading from geranyl diphosphate to the enzyme-bound, tertiary allylic intermediate linalyl diphosphate, which then cyclizes to the various monoterpene skeletons. X-ray crystal structures of these enzymes complexed with suitable analogues of the substrate and intermediate could provide a clearer view of this universal, but cryptic, step of monoterpenoid cyclase catalysis. Toward this end, the functionally inert analogues 2-fluorogeranyl diphosphate, (±)-2-fluorolinalyl diphosphate, and (3R)- and (3S)-homolinalyl diphosphates (2,6-dimethyl-2-vinyl-5-heptenyl diphosphates) were prepared, and compared to the previously described substrate analogue 3-azageranyl diphosphate (3-aza-2,3-dihydrogeranyl diphosphate) as inhibitors and potential crystallization aids with two representative monoterpenoid cyclases, (-)-limonene synthase and (+)-bornyl diphosphate synthase. Although these enantioselective synthases readily distinguished between (3R)- and (3S)-homolinalyl diphosphates, both of which were more effective inhibitors than was 3-azageranyl diphosphate, the fluorinated analogues proved to be the most potent competitive inhibitors and have recently yielded informative liganded structures with limonene synthase.     

阿诺碱受体及其亚型2调控因子研究进展

阿诺碱受体(RyR)是心肌细胞等可兴奋细胞中重要的Ca2+释放受体,在维持细胞的兴奋性和生理功能方面起重要作用.研究发现,RyR存在3个亚型,每个亚型都是由4个单体组成的四聚体,后者构成Ca2+释放通道.RyR的结构中有调控因子的结合位点,一些内源性调控因子可影响RyR的构型和Ca2+释放.结合作者的研究,就RyR的结构功能、RyR2的一些重要内源性调控因子及其调控机制做一简要综述.     

Roles and mechanisms of the CD38/cyclic adenosine diphosphate ribose/Ca2+ signaling pathway Wei W,Graeff R,Yue J简

Mobilization of intracellular Ca²⁺ stores is involved in many diverse cell functions, including: cell proliferation; differentiation; fertilization; muscle contraction; secretion of neurotransmitters, hormones and enzymes; and lymphocyte activation and proliferation. Cyclic adenosine diphosphate ribose (cADPR) is an endogenous Ca²⁺ mobilizing nucleotide present in many cell types and species, from plants to animals. cADPR is formed by ADP-ribosyl cyclases from nicotinamide adenine dinucleotide. The main ADP-ribosyl cyclase in mammals is CD38, a multi-functional enzyme and a type II membrane protein. It has been shown that many extracellular stimuli can induce cADPR production that leads to calcium release or influx, establishing cADPR as a second messenger. cADPR has been linked to a wide variety of cellular processes, but the molecular mechanisms regarding cADPR signaling remain elusive. The aim of this review is to summarize the CD38/cADPR/Ca²⁺ signaling pathway, focusing on the recent advances involving the mechanism and physiological functions of cADPR-mediated Ca²⁺ mobilization.     

The first prenylation step in hyperforin biosynthesis

Prenylation reactions contribute considerably to the diversity of natural products. Polyprenylated secondary metabolites include hyperforin which is both quantitatively and pharmacologically a major constituent of the medicinal plant Hypericum perforatum (St. John's wort). Cell cultures of the related species Hypericum calycinum were found to contain a prenyltransferase activity which is likely to catalyze the first prenylation step in hyperforin biosynthesis. The enzyme was soluble and dependent on a divalent cation, with Fe2+ leading to maximum activity (Km=3.8 mM). The preferred prenyl donor was DMAPP (Km=0.46 mM) and the preferred prenyl acceptor was phlorisobutyrophenone (Km=0.52 mM). A broad pH optimum from 6.5 to 8.5 and a temperature optimum from 35 to 40 degrees C were observed. The formation of hyperforins in H. calycinum cell cultures was preceded by an increase in dimethylallyltransferase activity, with the maximum specific activity being 3.6 microkat/kg protein.     

Detection of farnesyl diphosphate accumulation in yeast ERG9 mutants

A sensitive method was developed for measuring farnesyl diphosphate (FPP) accumulation in a mutant strain of Saccharomyces cerevisiae. The strain was blocked at squalene synthase (ERG9 gene) in the isoprenoid pathway and had the catalytic domain of the 3-hydroxy-3-methylglutaryl coenzyme A reductase gene integrated into the chromosome. It required ergosterol for growth and produced E,E-farnesol. The method was based on the isolation of FPP using the anion exchanger Macro Prep High Q and conversion of FPP to E,E-farnesol with alkaline phosphatase. Farnesol was measured using gas chromatography-mass spectrometry. Background farnesol in the cell-free extract was also retained by the anion exchanger, but was removed with repeated washing with methanol. Both 1M NaCl and 40% (v/v) methanol were required in the elution buffer to effectively elute FPP. The preparation of cell-free extract in Bis-Tris propane/HCl, pH 7, buffer containing 0.025% (w/v) Triton X-100 and 15 mM MgCl(2) provided optimum conditions for the stabilization of FPP.