Heteroaromatic quinols 4-(benzothiazol-2-yl)-4-hydroxycyclohexa-2,5-dienone (1) and 4-(1-benzenesulfonyl-1H-indol-2-yl)-4-hydroxycyclohexa-2,5-dienone (2) exhibit potent and selective antitumor activity against colon, renal, and breast carcinoma cell lines in vitro (GI 50 < 500 nmol/L). In vivo growth inhibition of renal, colon, and breast xenografts has been observed. Profound G 2 -M cell cycle block accompanied down-regulation of cdk1 gene transcription was corroborated by decreased CDK1 protein expression following treatment of HCT 116 cells with growth inhibitory concentrations of 1 or 2. The chemical structure of the quinol pharmacophore 4-(hydroxycyclohexa-2,5-dienone) suggested that these novel agents would readily react with nucleophiles in a double Michael (B-carbon) addition. Indeed, COMPARE analysis within the National Cancer Institute database revealed a number of chemically related quinone derivatives that could potentially react with sulfur nucleophiles in a similar manner and suggested that thioredoxin/ thioredoxin reductase signal transduction could be a putative target. Molecular modeling predicted covalent irreversible binding between quinol analogues and cysteine residues 32 and 35 of thioredoxin, thereby inhibiting enzyme activity. Binding has been confirmed, via mass spectrometry, between reduced human thioredoxin and 1. Microarray analyses of untreated HCT 116 cells and those exposed to either 1 (1 Mmol/L) or 2 (500 nmol/L and 1 Mmol/L) determined that of z z10,000 cancer-related genes, expression of thioredoxin reductase was up-regulated > >3-fold. Furthermore, quinols 1 and 2 inhibited insulin reduction, catalyzed by thioredoxin/thioredoxin reductase signaling in a dose-dependent manner (IC 50 < < 6 Mmol/L). Results are consistent with a mechanism of action of novel antitumor quinols involving inhibition of the small redox protein thioredoxin. (Cancer Res 2005; 65(9): 3911-9)
We report the use of a proteomic strategy to identify hitherto unknown substrates for mammalian protein L-isoaspartate O-methyltransferase. This methyltransferase initiates the repair of isoaspartyl residues in aged or stress-damaged proteins in vivo. Tissues from mice lacking the methyltransferase (Pcmt1 ؊/؊ ) accumulate more isoaspartyl residues than their wild-type littermates, with the most "damaged" residues arising in the brain. To identify the proteins containing these residues, brain homogenates from Pcmt1 ؊/؊ mice were methylated by exogenous repair enzyme and the radiolabeled methyl donor S-adenosyl-[methyl-3 H]methionine. Methylated proteins in the homogenates were resolved by both one-dimensional and twodimensional electrophoresis, and methyltransferase substrates were identified by their increased radiolabeling when isolated from Pcmt1 ؊/؊ animals compared with Pcmt1 ؉/؉ littermates.Mass spectrometric analyses of these isolated brain proteins reveal for the first time that microtubule-associated protein-2, calreticulin, clathrin light chains a and b, ubiquitin carboxylterminal hydrolase L1, phosphatidylethanolamine-binding protein, stathmin, -synuclein, and ␣-synuclein, are all substrates for the L-isoaspartate methyltransferase in vivo. Our methodology for methyltransferase substrate identification was further supplemented by demonstrating that one of these methyltransferase targets, microtubule-associated protein-2, could be radiolabeled within Pcmt1 ؊/؊ brain extracts using radioactive methyl donor and exogenous methyltransferase enzyme and then specifically immunoprecipitated with microtubule-associated protein-2 antibodies to recover co-localized protein with radioactivity. We comment on the functional significance of accumulation of relatively high levels of isoaspartate within these methyltransferase targets in the context of the histological and phenotypical changes associated with the methyltransferase knock-out mice.
Proteases play an important role in the programme of cell death by apoptosis but little is known of the substrates cleaved, particularly in constitutive models of this type of cell death. Neutrophils spontaneously undergo apoptosis in culture without requiring external stimuli. During this process we found biochemical and immunochemical evidence for the cleavage of membrane-associated actin, a component of the cytoskeleton that links polymerized actin to the plasma membrane. Cleavage occurred at a single site at the N-terminus, between residues Val43-Met44, a site devoid of a consensus motif for cleavage by cysteine proteases of the interleukin-1beta-converting enzyme (ICE)-family. Whereas actin cleavage and nuclear/cell surface markers of apoptosis were co-ordinately diminished by zVAD-fmk, an inhibitor of the ICE-like family of proteases, only acetyl-leucyl-leucylnormethional, an inhibitor of calpains, was capable of completely inhibiting actin cleavage. Our results suggest that actin is not a direct substrate for the ICE-like family of proteases. By disabling the cytoskeleton, actin cleavage may be an important component in the capacity of apoptosis to reduce the injurious potential of neutrophils.
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