PubMed · 6807858
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H Gordan. 1982. Herpes simplex.. https://doi.org/10.1111/j.1365-4362.1982.tb02082.x
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Proteasomes degrade endogenous proteins in the cytosol. The potential contribution of the proteasome to the effect of flanking sequences on the presentation of an antigenic epitope presented by the major histocompatibility complex class I allele Ld was studied. Peptides generated in cells from minigenes coding for peptides of 17- and 19-amino acid length were compared with the in vitro 20S proteasome degradation products of the respective synthetic peptides. The quality of generated peptides was independent of ubiquitination. In vivo and in vitro processing products were indistinguishable with respect to peptide size and abundance. Altering the neighboring sequence substantially improved the yield of the final antigenic nonapeptide by 20S proteasome cleavage. These results suggest that, in addition to the presence of major histocompatibility complex class I allelic motifs, the cleavage preference of the proteasome can define the antigenic potential of a protein.
4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a potent tobacco-specific carcinogen, has been demonstrated to induce lung tumors in animals and is suspected to be a human carcinogen. Cytochromes P450 are the major enzymes responsible for the activation of NNK in microsomes from the lung and liver of rat and mouse, as well as human liver. The present study investigated the enzymes responsible for the metabolic activation of NNK in human lung microsomes. In the presence of a NADPH-generating system, the formation of keto aldehyde and keto alcohol (alpha-hydroxylation products, measured together), keto acid, hydroxy acid, and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol was observed in human lung microsomes. Carbon monoxide (90%) decreased the rate of NNK oxidation by 5-49%, depending on the human lung microsomal samples analyzed. Coumarin decreased the oxidation of NNK by 9-34%, and an antibody against human P450 2A6 decreased the metabolism of NNK by 8-37%, suggesting the involvement of P450 2A6 in NNK oxidation. alpha-Napthoflavone inhibited NNK oxidation by 6-26%, possibly due to the inhibition of P450 1A1. P450 1A1-expressed microsomes catalyzed the formation of keto aldehyde and keto alcohol, exhibiting Km values of 1400 microM and 371 microM, respectively. In the absence of NADPH, NNK metabolism resulted in the formation of keto acid, keto aldehyde, and keto alcohol, and the activities in different lung samples were decreased by indomethacin (100 microM; cyclooxygenase inhibitor) or nordihydroguaiaretic acid (100 microM; lipoxygenase inhibitor) by 0-27% or 30-66%, respectively. The addition of arachidonic acid (10-100 microM) increased the rate of the formation of keto aldehyde and keto alcohol approximately 2-fold but inhibited the formation of keto acid. Soybean lipoxygenase increased the rate of formation of keto aldehyde and keto alcohol in a concentration-dependent manner. The increased rate in NNK oxidation by arachidonic acid or lipoxygenase was inhibited completely by nordihydroguaiaretic acid. Catalase, thiourea, and conjugated linoleic acid decreased the rate of NNK oxidation by 47, 20, and 45%, respectively. tert-Butyl-hydroperoxide, cumene hydroperoxide, and hydrogen peroxide increased the rate of formation of keto aldehyde and keto alcohol by 210, 40, and 50%, respectively. The results suggest that P450 enzymes are only partially responsible for the activation of NNK in human lung microsomes, and P450 2A6 or a P450 2A6-related enzyme seems to be involved in the activation. Furthermore, lipoxygenase and lipid hydroxperoxides may play important roles in the oxidation of NNK in human lung microsomes.
Monoaldehyde allosteric effectors of hemoglobin were designed, using molecular modeling software (GRID), to form a Schiff base adduct with the Val 1 alpha N-terminal nitrogens and interact via a salt bridge with Arg 141 alpha of the opposite subunit. The designed molecules were synthesized if not available. It was envisioned that the molecules, which are aldehyde acids, would produce a high-affinity hemoglobin with potential interest as antisickling agents similar to other aldehyde acids reported earlier. X-ray crystallographic analysis indicated that the aldehyde acids did bind as modeled de novo in symmetry-related pairs to the alpha subunit N-terminal nitrogens. However, oxygen equilibrium curves run on solutions obtained from T- (tense) state hemoglobin crystals of reacted effector molecules produced low-affinity hemoglobins. The shift in the allosteric equilibrium was opposite to that expected. We conclude that the observed shift in allosteric equilibrium was due to the acid group on the monoaldehyde aromatic ring that forms a salt bridge with the guanidinium ion of Arg 141 alpha on the opposite subunit. This added constraint to the T-state structure that ties two subunits across the molecular symmetry axis shifts the equilibrium further toward the T-state. We tested this idea by comparing aldehydes that form Schiff base interactions with the same Val 1 alpha residues but do not interact across the dimer subunit symmetry axis (a new one in this study with no acid group and others that have had determined crystal structures). The latter aldehydes shift the allosteric equilibrium toward the R-state. A hypothesis to predict the direction in shift of the allosteric equilibrium is made and indicates that it is not exclusively where the molecule binds but how it interacts with the protein to stabilize or destabilize the T- (tense) allosteric state.