ADP-Ribosylation of Highly Purified Rat Brain Mitochondria

Highly purified synaptic and nonsynaptic mitochondria were prepared from rat brain, and their ADP-ribosyl transferase and NAD glycohydrolase activities were investigated. Data show that there is no significant difference in ADP-ribosyl transferase activity between these two types of subcellular preparations. However, NAD glycohydrolase activity appeared to be much higher in nonsynaptic mitochondria. The specific activity of both enzymes was investigated in the presence of the inhibitor nicotinamide or its analogue 3-aminobenzamide or other adenine nucleotides, such as ATP or ADP-ribose. The inhibitory effect of nicotinamide or 3-aminobenzamide on ADP-ribosyl transferase appears rather weak compared with their effect on NAD glycohydrolase activity. However, ADP-ribose and ATP appeared more effective in inhibiting ADP-ribosyl transferase. Our results provide evidence for the existence of ADP-ribosyl transferase activity in rat brain mitochondria. When NAD glycohydrolase was inhibited totally by nicotinamide, the transfer of ADP-ribose from NAD to mitochondrial proteins still occurred. The chain length determinations show that the linkage of ADP-ribose to mitochondrial proteins is oligomeric.     

Mapping the enzymatic active site of Pseudomonas aeruginosa exotoxin A

Pseudomonas aeruginosa exotoxin A is representative of a class of enzymes, the monoADP-ribosyl, which catalyze the covalent transfer of an ADP-ribose moiety of NAD⁺ to a target substrate. Availability of the three-dimensional structure of exotoxin A provides the opportunity for mapping substrate binding sites and suggesting which amino acid residues may be involved in catalysis. Data from several sources have been combined to develop a proposal for the NAD⁺ binding site of exotoxin A: the binding of NAD⁺ fragments adenosine, AMP, and ADP have been delineated crystallographically to 6.0, 6.0, and 2.7 Å, respectively; significant sequence homology spanning 60 residues has been found between exotoxin A and diphtheria toxin, which has the identical enzymatic activity; iodination of exotoxin A, under conditions in which only tyrosine 481 is iodinated in the enzymatic domain, abolishes ADP-ribosyl transferase activity.     

DeoxyNAD and deoxyADP-ribosylation of proteins

Recently, two deoxyribose analogs of NAD⁺ (2-deoxy and 3-deoxyNAD⁺) have been synthesized and purified in this laboratory. Whereas 2-deoxyNAD⁺ was an efficient substrate for arg-specific mon(ADP-ribosyl) transferases, it was not a substrate for poly(ADP-ribose) polymerase (PARP). Instead, it was a non-competitive inhibitor of NAD⁺ in the ADP-ribose polymerization reaction catalyzed by PARP. Thus, 2-deoxyNAD⁺ has been utilized to distinguish between mono(ADP-ribose) and poly(ADP-ribose) acceptor proteins. 2-deoxyNAD⁺ has also been used to characterize the arg-specific mono(2-deoxyADP-ribosyl)ation reaction of PARP with cholera toxin or avian mono(ADP-ribosyl)transferase. By contrast, 3-deoxyNAD⁺ can effectively be utilized as a substrate by PARP. However, while the estimated Km and Kcat of polymerization with 3-deoxyNAD⁺ can were 20 M and 0.11 moles/sec, the Km and Kcat with NAD⁺ as a substrate were 59 M and 1.29 moles/sec, respectively. Determination of the average size of 3-deoxyADP-ribose polymers indicated that chains no larger than four residues are synthesized with this substrate. Thus, the utilization of 3-deoxyNAD⁺ has facilitated the electrophoretic identification of poly(ADP-ribose) acceptor proteins in mammalian chromatin.     

Telomere elongation by a mutant tankyrase 1 without TRF1 poly(ADP-ribosyl)ation

Telomeres are the capping structures of the eukaryotic chromosome ends. Tankyrase 1 is a poly(ADP-ribose) polymerase that elongates telomeres in a telomerase-dependent manner. This function of tankyrase 1 is mediated by down-regulation of TRF1, a negative regulator of telomere access to telomerase. Namely, tankyrase 1 poly(ADP-ribosyl)ates (PARsylates) TRF1, which in turn dissociates TRF1 from telomeres. The resulting telomeres become better substrates for telomerase-mediated DNA extension. Tankyrase 1 has five independent TRF1 binding sites, ARC (ANK repeat cluster) I to V. Among them, the most C-terminal ARC V is required for TRF1 PARsylation and its release from telomeres. By contrast, functional significance of other four ARCs remains elusive. In this study, we generated a mutant tankyrase 1 that had inactive ARC IV and lacked ARC V but elongated telomeres without TRF1 PARsylation. Consistent with the failure in PARsylation, this mutant only marginally released TRF1 from telomeres. Still, it decreased telomere binding of POT1, a downstream effector of TRF1-mediated telomere length control, and elongated the telomeric 3'-overhang as the wild-type tankyrase 1 did. Thus even without TRF1 PARsylation, this mutant tankyrase 1 seemed to loosen the closed structure of the telomeric heterochromatin. These findings suggest a new role for multiple ARCs in telomere extension by tankyrase 1.     

Structure of Cinaciguat (BAY 58–2667) Bound to Nostoc H-NOX Domain Reveals Insights into Heme-mimetic Activation of the Soluble Guanylyl Cyclase

Heme is a vital molecule for all life forms with heme being capable of assisting in catalysis, binding ligands, and undergoing redox changes. Heme-related dysfunction can lead to cardiovascular diseases with the oxidation of the heme of soluble guanylyl cyclase (sGC) critically implicated in some of these cardiovascular diseases. sGC, the main nitric oxide (NO) receptor, stimulates second messenger cGMP production, whereas reactive oxygen species are known to scavenge NO and oxidize/inactivate the heme leading to sGC degradation. This vulnerability of NO-heme signaling to oxidative stress led to the discovery of an NO-independent activator of sGC, cinaciguat (BAY 58–2667), which is a candidate drug in clinical trials to treat acute decompensated heart failure. Here, we present crystallographic and mutagenesis data that reveal the mode of action of BAY 58–2667. The 2.3-Å resolution structure of BAY 58–2667 bound to a heme NO and oxygen binding domain (H-NOX) from Nostoc homologous to that of sGC reveals that the trifurcated BAY 58–2667 molecule has displaced the heme and acts as a heme mimetic. Carboxylate groups of BAY 58–2667 make interactions similar to the heme-propionate groups, whereas its hydrophobic phenyl ring linker folds up within the heme cavity in a planar-like fashion. BAY 58–2667 binding causes a rotation of the αF helix away from the heme pocket, as this helix is normally held in place via the inhibitory His¹⁰⁵–heme covalent bond. The structure provides insights into how BAY 58–2667 binds and activates sGC to rescue heme-NO dysfunction in cardiovascular diseases.     

GM3对重建Gs与AC偶联功能的影响

将神经节苷脂ＧＭ３（Ｍｏｎｏｓｉａｌｏｇａｎｇｌｉｏｓｉｄｅ－ＧＭ３）通过保温法掺入到含激活型Ｇ蛋白（ＳｔｉｍｕｌａｔｏｒｙＧＴＰ－ｂｉｎｄｉｎｇｐｒｏｔｅｉｎ,Ｇｓ）与腺苷酸环化酶（ＡｄｅｎｙｌｙｌＣｙｃｌａｓｅ,ＡＣ）的脂酶体中,研究了ＧＭ３对Ｇｓ和ＡＣ偶联功能的影响。实验结果表明,在４－１０μｍｏｌ／Ｌ浓度范围的ＧＭ３增加ＡＣ的基础活力;在高于４μｍｏｌ／Ｌ时,ＧＭ３可显著抑制Ｇｓ激活ＡＣ的能力;而在ＧＭ３浓度高于１００μｍｏｌ／Ｌ的条件下,Ｇｓ结合ＧＴＰγＳ（Ｇｕａｎｏｓｉｎｅ５＇－Ｏ－（３－ｔｈｉｏｔｒｉｐｈｏｓｐｈａｔｅ））的活力受到明显抑制。随外源ＧＭ３浓度的增加,ＧＭ３对Ｇｓ激活ＡＣ的能力与对ＡＣ基础活力的影响似乎并不完全一致。这些结果提示,Ｇｓ与ＡＣ的解偶联对较低浓度的ＧＭ３的影响更加敏感。用荧光探剂ＭＣ５４０标记脂酶体,测量其荧光光谱的结果显示,随着ＧＭ３浓度增加,ＭＣ５４０的荧光强度增强,这说明外源性的ＧＭ３的掺入使膜脂质分子头部的堆积变得更加疏松。这可能提示,ＧＭ３介导的膜脂物理状态的变化是调节Ｇｓ与ＡＣ偶联功能的重要因素之一。     

Measurement of adenylyl cyclase by separating cyclic AMP on silica gel thin-layer chromatography

A procedure for isolation of cyclic AMP (cAMP) by thin-layer chromatography on silica gel is described. One-dimensional ascending chromatograms were developed using [H(2)O/C(2)H(5)OH/NH(4)HCO(3) (30%:70%:0.2M)] as the mobile phase. This procedure separated [32P]cAMP from other radioactive metabolites of [32P]ATP in up to 19 samples on one sheet (20 x 10 cm) over 40-60 min at room temperature (21 degrees C). This simple and rapid isolation method provides a novel and convenient technique for the assay of adenylyl cyclase.     

Light inhibits rifampicin inactivation and reduces rifampicin resistance due to a cloned mycobacterial ADP-ribosylation gene

Rifampicin is a principal drug used to combat infections by mycobacteria and related organisms. Most strains of Mycobacterium are able to inactivate this antibiotic by ribosylation via an ADP-ribosylated intermediate. We found that this inactivation was inhibited by light at levels similar to those prevailing in laboratory environments. Rifampicin resistance arising from the cloned ADP-ribosyl transferase was also greatly diminished at these light levels. The cloned Rhodococcus equi monooxygenase which inactivates this antibiotic by a different mechanism was, in contrast, not inhibited by light.     

CO2/HCO3?- and Calcium-regulated Soluble Adenylyl Cyclase as a Physiological ATP Sensor

The second messenger molecule cAMP is integral for many physiological processes. In mammalian cells, cAMP can be generated from hormone- and G protein-regulated transmembrane adenylyl cyclases or via the widely expressed and structurally and biochemically distinct enzyme soluble adenylyl cyclase (sAC). sAC activity is uniquely stimulated by bicarbonate ions, and in cells, sAC functions as a physiological carbon dioxide, bicarbonate, and pH sensor. sAC activity is also stimulated by calcium, and its affinity for its substrate ATP suggests that it may be sensitive to physiologically relevant fluctuations in intracellular ATP. We demonstrate here that sAC can function as a cellular ATP sensor. In cells, sAC-generated cAMP reflects alterations in intracellular ATP that do not affect transmembrane AC-generated cAMP. In β cells of the pancreas, glucose metabolism generates ATP, which corresponds to an increase in cAMP, and we show here that sAC is responsible for an ATP-dependent cAMP increase. Glucose metabolism also elicits insulin secretion, and we further show that sAC is necessary for normal glucose-stimulated insulin secretion in vitro and in vivo.     

ARTD1 (PARP1) activation and NAD+ in DNA repair and cell death

Nicotinamide adenine dinucleotide, NAD⁺, is a small metabolite coenzyme that is essential for the progress of crucial cellular pathways including glycolysis, the tricarboxylic acid cycle (TCA) and mitochondrial respiration. These processes consume and produce both oxidative and reduced forms of NAD (NAD⁺ and NADH). NAD⁺ is also important for ADP(ribosyl)ation reactions mediated by the ADP-ribosyltransferase enzymes (ARTDs) or deacetylation reactions catalyzed by the sirtuins (SIRTs) which use NAD⁺ as a substrate. In this review, we highlight the significance of NAD⁺ catabolism in DNA repair and cell death through its utilization by ARTDs and SIRTs. We summarize the current findings on the involvement of ARTD1 activity in DNA repair and most specifically its involvement in the trigger of cell death mediated by ARTD1 activation and energy depletion. By sharing the same substrate, the activities of ARTDs and SIRTs are tightly linked, are dependent on each other and are thereby involved in the same cellular processes that play an important role in cancer biology, inflammatory diseases and ischaemia/reperfusion.