The degradation of ␣II-and ␤II-spectrin during apoptosis in cultured human neuroblastoma SH-SY5Y cells was investigated. Immunofluorescent staining showed that the collapse of the cortical spectrin cytoskeleton is an early event following staurosporine challenge. This collapse correlated with the generation of a series of prominent spectrin breakdown products (BDPs) derived from both ␣II-and ␤II-subunits. Major C-terminal ␣II-spectrin BDPs were detected at Ϸ150, 145, and 120 kDa (␣II-BDP150, ␣II-BDP145, and ␣II-BDP120, respectively); major C-terminal ␤II-spectrin BDPs were at Ϸ110 and 85 kDa (␤II-BDP110 and ␤II-BDP85, respectively). N-terminal sequencing of the major fragments produced in vitro by caspase 3 revealed that ␣II-BDP150 and ␣II-BDP120 were generated by cleavages at DETD 1185 *S 1186 and DSLD 1478 *S 1479 , respectively. For ␤II-spectrin, a major caspase site was detected at DEVD 1457 *S 1458, and both ␤II-BDP110 and ␤II-BDP85 shared a common N-terminal sequence starting with Ser 1458 . An additional cleavage site near the C terminus, at ETVD 2146 *S 2147 , was found to account for ␤II-BDP85. Studies using specific caspase or calpain inhibitors indicate that the pattern of spectrin breakdown during apoptosis differs from that during non-apoptotic cell death. We postulate that in concert with calpain, caspase rapidly targets critical sites in both ␣II-and ␤II-spectrin and thereby initiates a rapid dissolution of the spectrin-actin cortical cytoskeleton with apoptosis.The importance of proteases in the expression of mammalian apoptosis has been the subject of many recent studies. The mammalian interleukin-1␤-converting enzyme (ICE) 1 -like protease family (renamed caspase (1) (5), and its deletion by gene knockout blocks neuronal death during brain development with consequential lethality (6). Besides the caspases, a second family of proteases implicated in the initiation and control of apoptosis are the calpains (7, 8), especially in several hematopoietic and neuronal cells (9 -12). The relationship between these two protease families, the consequences of each on their respective substrates and on cellular physiology, or the conditions under which each is activated remain poorly understood. While many proteins are cleaved during apoptosis, a prominent target of both calpain and caspase action is ␣II-spectrin, the major component of the cortical membrane skeleton. In neurons, calcium-activated calpain cleavage of ␣II-spectrin (non-erythroid ␣-spectrin or ␣-fodrin) accompanies N-methyl-D-aspartic acid receptor activation (13), 2 does not directly cause neuronal toxicity (7,15), and is postulated to be necessary for synaptic and neuronal plasticity (16 -18). Indeed, ␣II-spectrin cleavage by calpain appears to be a molecular mechanism by which skeletal plasticity can be enhanced without complete dissolution of the spectrin skeleton since calpain-mediated cleavage of ␣II-spectrin bestows calmodulin regulation on oligomeric spectrin-actin complexes, but does not dissociate them (unless ␤II-spectrin is also c...

The full-length, protein coding sequence for dehaloperoxidase was obtained using a reverse genetic approach and a cDNA library from marine worm Amphitrite ornata. The crystal structure of the dehaloperoxidase (DHP) was determined by the multiple isomorphous replacement method and was refined at 1.8-Å resolution. The enzyme fold is that of the globin family and, together with the amino acid sequence information, indicates that the enzyme evolved from an ancient oxygen carrier. The peroxidase activity of DHP arose mainly through changes in the positions of the proximal and distal histidines relative to those seen in globins. The structure of a complex of DHP with 4-iodophenol is also reported, and it shows that in contrast to larger heme peroxidases DHP binds organic substrates in the distal cavity. The binding is facilitated by the histidine swinging in and out of the cavity. The modeled position of the oxygen atom bound to the heme suggests that the enzymatic reaction proceeds via direct attack of the oxygen atom on the carbon atom bound to the halogen atom.Polychlorinated phenols and other polychlorinated aromatics of anthropogenic origin have been widely dispersed and constitute significant environmental problems. It is less known that bromoaromatics of biotic origin are also widespread and secreted as chemical warfare by a number of marine organisms. Dehalogenating enzymes are used as the first line of defense against these toxicants by organisms that live in such contaminated environments (1). We have recently discovered and characterized by a number of techniques (2-4) an enzyme with a novel function, dehaloperoxidase (DHP).1 DHP is isolated from Amphitrite ornata, a terebellid polychaete. This species does not produce halogenated compounds itself but usually co-habits estuarine mud flats with other polychaete worms, such as Notomastus lobatus, and hemichordata such as Saccoglossus kowalewskyi, which secrete large quantities of brominated aromatics and other halometabolites as repellents (5). The levels of DHP are very high as it represents approximately 3% of the soluble protein in crude extracts of A. ornata. The enzyme catalyzes the oxidative dehalogenation of polyhalogenated phenols in the presence of hydrogen peroxide at a rate at least 10 times faster than all known halohydrolases of bacterial origin, according to Reaction 1.The oxidative potential of hydrogen peroxide likely allows for the unusually high rate of this reaction as well as for the unique ability of DHP to dehalogenate fluorophenols. The enzyme has activity toward substrates with different numbers and positions of halogen substituents (2).The binding of oxygen and peroxide ligands and their activation are due to the presence of heme in a variety of oxygen carriers and enzymes. This is also true for DHP, which contains one heme per subunit (3) and a histidine as the proximal iron ligand (4). The propensity of peroxidases (and oxygenases, which tend to have a cysteinate proximal ligand) to cleave the oxygen-oxygen bond and form a high vale...

No abstract

We have previously demonstrated cleavage of ␣-spectrin by caspase-3 and calpain during apoptosis in SH-SY5Y neuroblastoma cells (Nath, R., Raser, K. J., Stafford, D., Hajimohammadreza, I., Posner, A., Allen, H., Talanian, R. V., Yuen, P., Gilbertsen, R. B., and Wang, K. K. (1996) Biochem. J. 319, 683-690). We demonstrate here that calcium/calmodulin-dependent protein kinase IV (CaMK IV) is cleaved during apoptosis by caspase-3 and calpain. We challenged SH-SY5Y cells with the proapoptotic agent thapsigargin. Western blot analysis revealed major CaMK IV breakdown products of 40, 38, and 33 kDa. Digestion of control SH-SY5Y lysate with purified caspase-3 produced a 38-kDa CaMK IV fragment; digestion with purified calpain produced a major fragment of 40 kDa. Pretreatment with carbobenzoxyAsp-CH 2 OC(O)-2,6-dichlorobenzene or Z-Val-Ala-Aspfluoromethylketone was able to block the caspase-3-mediated production of the 38-kDa fragment both in situ and in vitro. Calpain inhibitor II similarly blocked formation of the calpain-mediated 40-kDa fragment both in situ and in vitro. Digestion of recombinant CaMK IV by other caspase family members revealed that only caspase-3 produces a fragmentation pattern consistent to that seen in situ. The major caspase-3 and calpain cleavage sites are respectively identified as PAPD 176 *A and CG 201 *A, both within the CaMK IV catalytic domain. Furthermore, calmodulin-stimulated protein kinase activity decreases within 6 h in thapsigargin-treated SH-SY5Y. The loss of activity precedes cell death.

No abstract