M Moroni

Oxidative modification of aldose reductase induced by copper ion. Definition of the metal-protein interaction mechanism.

Aldose reductase (ALR2) is susceptible to oxidative inactivation by copper ion. The mechanism underlying the reversible modification of ALR2 was studied by mass spectrometry, circular dichroism, and molecular modeling approaches on the enzyme purified from bovine lens and on wild type and mutant recombinant forms of the human placental and rat lens ALR2. Two equivalents of copper ion were required to inactivate ALR2: one remained weakly bound to the oxidized protein whereas the other was strongly retained by the inactive enzyme. Cys303 appeared to be the essential residue for enzyme inactivation, because the human C303S mutant was the only enzyme form tested that was not inactivated by copper treatment. The final products of human and bovine ALR2 oxidation contained the intramolecular disulfide bond Cys298-Cys303. However, a Cys80-Cys303 disulfide could also be formed. Evidence for an intramolecular rearrangement of the Cys80-Cys303 disulfide to the more stable product Cys298-Cys303 is provided. Molecular modeling of the holoenzyme supports the observed copper sequestration as well as the generation of the Cys80-Cys303disulfide. However, no evidence of conditions favoring the formation of the Cys298-Cys303 disulfide was observed. Our proposal is that the generation of the Cys298-Cys303 disulfide, either directly or by rearrangement of the Cys80-Cys303 disulfide, may be induced by the release of the cofactor from ALR2 undergoing oxidation. The occurrence of a less interactive site for the cofactor would also provide the rationale for the lack of activity of the disulfide enzyme forms.

Post-transcriptional control regulates transforming growth factor alpha in the human carcinoma KB cell line.

Expression of epidermal growth factor receptor (EGF-R) antisense RNA results in a drastic reduction of EGF-R levels in the human carcinoma KB cell line and induces a reversion of their transformed phenotype (Moroni, M. C., Willingham, M. C., and Beguinot, L. (1992) J. Biol. Chem. 267, 2714-2722). We used parental and EGF-R antisense KB clones as a genetic system to study, in the same cell line, the role of transforming growth factor α (TGF-α) in the establishment and maintenance of the transformed phenotype. KB cells produce TGF-α mRNA, and their conditioned medium is able to sustain growth of antisense cells, mimicking the effect of exogenous EGF or TGF-α. In antisense cells there is a marked reduction of TGF-α mRNA steady-state levels. In addition, the decrease in TGF-α parallels the levels of residual EGF-R in the various antisense clones, indicating a direct correlation between receptors and growth factor levels. The addition of exogenous TGF-α (10 ng/ml) to antisense clones induces TGF-α levels. The half-life of TGF-α mRNA is 40-60 min in antisense cells and more than 8 h in parental KB cells, as determined by actinomycin D decay curves. This result indicates a predominant regulation of TGF-α mRNA at the post-transcriptional level. Nuclear run-on experiments show that there is only a marginal effect at the transcriptional level. We conclude that the autocrine loop responsible for the transformed phenotype of the human carcinoma KB cell line is dependent on both elevated levels of EGF-R and the presence of TGF-α. In addition, TGF-α is able to induce its own mRNA via a signal due to activation of the EGF-R acting predominantly at the post-transcriptional level.