Tyrosyl DNA phosphodiesterase I (Tdp1) is a member of the phospholipase D superfamily and hydrolyzes 3′phospho-DNA adducts via two conserved catalytic histidines, one acting as the lead nucleophile and the second as a general acid/base. Substitution of the second histidine specifically to arginine contributes to the neurodegenerative disease SCAN1. We investigated the catalytic role of this histidine in the yeast protein (His432) using a combination of X-ray crystallography, biochemistry, yeast genetics and theoretical chemistry. The structures of wild type Tdp1 and His432Arg both show a phosphorylated form of the nucleophilic histidine that is not observed in the structure of His432Asn. The phosphohistidine is stabilized in the His432Arg structure by the guanidinium group that also restricts access of a nucleophilic water molecule to the Tdp1-DNA intermediate. Biochemical analyses confirm that His432Arg forms an observable and unique Tdp1-DNA adduct during catalysis. Substitution of His432 by Lys does not affect catalytic activity or yeast phenotype, but substitution with Asn, Gln, Leu, Ala, Ser and Thr all result in severely compromised enzymes and Top1-camptothecin dependent lethality. Surprisingly, His432Asn did not show a stable covalent Tdp1-DNA intermediate which suggests another catalytic defect. Theoretical calculations revealed that the defect resides in the nucleophilic histidine and that the pKa of this histidine is crucially dependent upon the second histidine and the incoming phosphate of the substrate. This represents a unique example of substrate-activated catalysis that applies to the entire phospholipase D superfamily.

No abstract

Abstract-Angiotensin II and the arachidonic acid metabolite derived via cytochrome P450 20-hydroxyeicostetraenoic acid promote vasoconstriction and vascular smooth muscle cell (VSMC) proliferation. This study was conducted to determine if 20-hydroxyeicostetraenoic acid contributes to angiotensin II-induced neointimal formation in ballooninjured rat carotid artery. In anesthetized rats, the drugs were infused into the clamped segment of the injured right common carotid artery for 60 minutes. The drug solution and catheter were withdrawn, the common carotid artery was ligated, and blood flow was restored. Exposure of the injured artery to angiotensin II (200 nmol/L) or arachidonic acid (10 mol/L) increased neointimal thickening at day 14 (intima/media ratio 0.71Ϯ0.14 with vehicle versus 1.65Ϯ0.10 with angiotensin II or 1.31Ϯ0.13 with arachidonic acid; PϽ0.05). Cytochrome P450 4A1 antisense, but not scrambled, oligodeoxynucleotide (100 nmol/L) reduced angiotensin II-induced or arachidonic acid-induced neointimal thickening (intima/media ratio 0.90Ϯ0.07 for angiotensin II and 0.95Ϯ0.06 for arachidonic acid). 20-hydroxyeicostetraenoic acid (0.5 mol/L) also increased neointimal thickening of injured artery (intima/media ratio 1.15Ϯ0.03); this was not altered by cytochrome P450 4A1 antisense oligodeoxynucleotide. Angiotensin II, arachidonic acid, and 20-hydroxyeicostetraenoic acid also induced the expression of cytochrome P450 4A and increased the number of CD45-positive cells; the latter effect of angiotensin II and arachidonic acid, but not 20-hydroxyeicostetraenoic acid, was diminished by cytochrome P450 4A1 antisense oligodeoxynucleotide. These data suggest that arachidonic acid metabolites derived via cytochrome P450 4A, most likely 20-hydroxyeicostetraenoic acid, mediate angiotensin II-induced neointimal thickening in injured rat carotid artery. Key Words: angiotensin II Ⅲ arachidonic acids Ⅲ balloon injury Ⅲ cytochrome p450 Ⅲ vascular smooth muscle A ngiotensin II (Ang II), a biologically active octapeptide generated by the renin-angiotensin system, contributes to the regulation of blood pressure by maintaining vascular tone and salt and water balance. High levels of Ang II promote vascular smooth muscle cell (VSMC) hypertrophy, migration, and delayed hyperplasia, and cause inflammation, neointimal formation, and hypertension. 1,2 Growth factors such as Ang II promote activation of transcription factors (nuclear factor B and activating protein-1) and proinflammatory genes (cytokines, interleukins), upregulation of adhesion molecules (intercellular adhesion molecule, vascular cell adhesion molecule), stimulation of chemokine production (monocyte chemoattractant protein-1), and their receptors, and recruitment of inflammatory cells (monocytes, macrophages), all of which are critical processes involved in vascular inflammation and injury. 2,3 Ang II also activates cytosolic phospholipase A 2 , which catalyzes the hydrolysis of phospholipids, leading to release of arachidonic acid (AA). AA is subsequently metabolize...

Cytosolic phospholipase A 2 (cPLA 2 ) is activated and translocated to the nuclear envelope by various vasoactive agents, including norepinephrine (NE), and releases arachidonic acid (AA) from tissue phospholipids. We previously demonstrated that NE-induced cPLA 2 translocation to the nuclear envelope is mediated via its phosphorylation by calcium/calmodulindependent kinase-II in rabbit vascular smooth muscle cells (VSMCs). Cytoskeletal structures actin and microtubule filaments have been implicated in the trafficking of proteins to various cellular sites. This study was conducted to investigate the contribution of actin and microtubule filaments to cPLA 2 translocation to the nuclear envelope and its activation by NE in rabbit VSMCs. NE (10 M) caused cPLA 2 translocation to the nuclear envelope as determined by immunofluorescence. Cytochalasin D (CD; 0.5 M) and latrunculin A (LA; 0.5 M) that disrupted actin filaments, blocked cPLA 2 translocation elicited by NE. On the other hand, disruption of microtubule filaments by 10 M colchicine did not block NE-induced cPLA 2 translocation to the nuclear envelope. CD and LA did not inhibit NE-induced increase in cytosolic calcium and cPLA 2 activity, determined from the hydrolysis of L-1-[14 C]arachidonyl phosphatidylcholine and release of AA. Coimmunoprecipitation studies showed an association of actin with cPLA 2 , which was not altered by CD or LA. Far-Western analysis showed that cPLA 2 interacts directly with actin. Our data suggest that NEinduced cPLA 2 translocation to the nuclear envelope requires an intact actin but not microtubule filaments and that cPLA 2 phosphorylation and activation and AA release are independent of its translocation to the nuclear envelope in rabbit VSMCs.

No abstract