Biochemistry
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Bizelesin, a bifunctional DNA minor groove alkylating agent, inhibits both cellular and viral (SV40) DNA replication in whole cells. Bizelesin inhibition of SV40 DNA replication was analyzed in SV40-infected cells, using two-dimensional (2D) neutral agarose gel electrophoresis, and in a cell-free SV40 DNA replication assay. Within 1 h of bizelesin addition to infected cells, a similar rapid decrease in both the level of SV40 replication intermediates and replication activity was observed, indicating inhibition of initiation of SV40 DNA replication. ⋯ The level of in vitro replication of SV40 DNA also was reduced when extracts from bizelesin-treated HeLa cells were used. This effect was not dependent upon the formation of bizelesin covalent bonds with the template DNA. Mixing experiments, using extracts from control and bizelesin-treated cells, indicated that reduced DNA replication competence was due to the presence of a trans-acting DNA replication inhibitor, rather than to decreased levels or inactivation of essential replication factor(s).
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In the presence of phospholipid vesicles and calcium ions, protein Z (PZ) serves as a cofactor for the inhibition of coagulation factor Xa by a plasma protein called PZ-dependent protease inhibitor (ZPI). To further characterize ZPI, its cDNA has been isolated and cloned from a human liver cDNA library. The ZPI cDNA is 2.44 kb in length and has a relatively long 5' region (466 nt) that contains six potential ATG translation start codons. ⋯ Thus, ZPI is likely the human homologue of rat rasp-1. Alignment of the amino acid sequence of ZPI with those of other serpins predicts that Y387 is the P(1) residue at the reactive center of the ZPI molecule. Consistent with this notion, rZPI(Y387A), an altered form of ZPI in which tyrosine 387 has been changed to alanine, lacks PZ-dependent factor Xa inhibitory activity.
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The amyloid precursor protein (APP) is proteolytically processed predominantly by alpha-secretase to release the ectodomain (sAPPalpha). In this study, we have addressed the cellular location of the constitutive alpha-secretase cleavage of endogenous APP in a neuronal cell line. Incubation of the neuroblastoma cell line IMR32 at 20 degrees C prevented the secretion into the medium of soluble wild-type APP cleaved by alpha-secretase as revealed by both immunoelectrophoretic blot analysis with a site-specific antibody and immunoprecipitation following metabolic labeling of the cells. ⋯ Parallel studies using an antibody that recognizes specifically the neoepitope revealed on soluble APP cleaved by beta-secretase indicated that this enzyme was acting intracellularly. alpha-Secretase is a zinc metalloproteinase susceptible to inhibition by hydroxamate-based compounds such as batimastat [Parvathy, S., et al. (1998) Biochemistry 37, 1680-1685]. Incubation of the cells with a cell-impermeant, biotinylated hydroxamate inhibitor inhibited the release of sAPPalpha by >92%, indicating that alpha-secretase is cleaving APP almost exclusively at the cell surface. The observation that alpha-secretase cleaves APP at the cell surface, while beta-secretase can act earlier in the secretory pathway within the neuronal cell line indicates that there must be strict control mechanisms in place to ensure that APP is normally cleaved primarily by alpha-secretase in the nonamyloidogenic pathway to produce the neuroprotective sAPPalpha.
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Comparative Study
Crystal structure of human D-dopachrome tautomerase, a homologue of macrophage migration inhibitory factor, at 1.54 A resolution.
D-Dopachrome tautomerase shares a low homologous amino acid sequence (33% homology) with the macrophage migration inhibitory factor (MIF) and possesses similar tautomerase activity as well. MIF is a cytokine involved in inflammatory reactions and immune responses. Whereas recent studies have identified MIF as a pituitary hormone and immunoregulator, much less is known about the structural basis of these physiological functions and the real significance of tautomerase activity. ⋯ A detailed comparison of these structures revealed significant differences in the environment around the potential active site, the intersubunit contacts, and charge distribution on the molecular surface. It can be concluded that these features are related to the physiological role and tautomerase activity of MIF and D-dopachrome tautomerase. The present structural study could be helpful for designing effective inhibitors that modulate immunoregulatory and hormone-like effects.
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Whether ultimately utilized as retinoic acid, retinal, or retinol, vitamin A is transported to the target cells as all-trans-retinol bound to retinol-binding protein (RBP). Circulating in the plasma, RBP itself is bound to transthyretin (TTR, previously referred to as thyroxine-binding prealbumin). In vitro one tetramer of TTR can bind two molecules of retinol-binding protein. ⋯ In addition, the structure reveals an interaction of the carboxy terminus of RBP at the protein-protein recognition interface. This interaction, which involves Leu-182 and Leu-183 of RBP, is consistent with the observation that naturally occurring truncated forms of the protein are more readily cleared from plasma than full-length RBP. Complex formation prevents extensive loss of RBP through glomerular filtration, and the loss of Leu-182 and Leu-183 would result in a decreased affinity of RBP for TTR.