PubMed · 7887217
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M W Hentze. 1994. Translational control by iron-responsive elements.. https://doi.org/10.1007/978-1-4615-2554-7_13
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Utilization of mRNAs containing iron-responsive elements (IREs) is modulated by iron-regulated RNA-binding proteins (iron regulatory proteins). We examine herein whether iron differentially affects translation of ferritin and mitochondrial aconitase (m-Acon) mRNAs because they contain a similar but not identical IRE in their 5'-untranslated regions. First, we demonstrate that m-Acon synthesis is iron-regulated in mammalian cells. In HL-60 cells, hemin (an iron source) stimulated m-Acon synthesis 3-fold after 4 h compared with cells treated with an iron chelator (Desferal). Furthermore, hemin stimulated m-Acon synthesis 2-4-fold in several cell lines. Second, we show that iron modulates the polysomal association of m-Acon mRNA. We observed m-Acon mRNA in both ribonucleoprotein and polyribosomal fractions of HL-60 cells. Hemin significantly increased the polyribosomal association and decreased the ribonucleoprotein abundance of m-Acon mRNA in HL-60 cells. Third, our results indicate that iron differentially regulates translation of m-Acon and ferritin mRNAs. A dose response to hemin in HL-60 cells elicited a 2-2.4-fold increase in m-Acon synthesis within 5 h compared with untreated cells, whereas ferritin synthesis was stimulated 20-100-fold. We conclude that iron modulates m-Acon synthesis at the translational level and that iron regulatory proteins appear to differentially affect translation of IRE-containing mRNAs.
The enzymes which are responsible for catalyzing sequential reactions in several metabolic pathways have been proposed to be highly organized in supramolecular complexes termed metabolons. However, the in situ existence of these weak complexes is difficult to demonstrate because many of them are dissociated during isolation due to dilution effects. Consequently, the metabolon concept is subject to controversy. A model system consisting of genetically prepared bienzymatic fusion proteins has been used to immobilize sequential metabolic enzymes in close proximity and to demonstrate possible kinetic advantages of metabolons. These experiments use the sequential Krebs TCA cycle enzymes from yeast mitochondrial malate dehydrogenase (MDH), citrate synthase (CS), and aconitase (ACO). Using the porcine high-definition structures of these three enzymes, we have performed computer-modeling studies in order to understand how the molecules may interact. Among the thousands of docking orientations we have tried, one was found to respond to the structural and experimental constraints from the results obtained with the yeast fusion proteins. Interestingly, this quinary structure model shows substantial interacting surface areas with spatial and electrostatic complementarities which make the complex thermodynamically stable. This structure also contains an unbroken electrostatically favorable channel connecting the active sites of ACO and CS, as well as the one previously reported between CS and MDH active sites. Charged amino acids which could be involved in interactions stabilizing the complex have been identified. This model will be used as the basis for further experimental work on the structure of the Krebs TCA cycle metabolon.
Prostate epithelial cells possess a uniquely limiting mitochondrial (m-) aconitase activity that minimizes their ability to oxidize citrate. These cells also possess uniquely high cellular and mitochondrial zinc levels. Correlations among zinc, citrate, and m-aconitase in prostate indicated that zinc might be an inhibitor of prostate m-aconitase activity and citrate oxidation. The present studies reveal that zinc at near physiological levels inhibited m-aconitase activity of mitochondrial sonicate preparations obtained from rat ventral prostate epithelial cells. Corresponding studies conducted with mitochondrial sonicates of rat kidney cells revealed that zinc also inhibited the kidney m-aconitase activity. However the inhibitory effect of zinc was more sensitive with the prostate m-aconitase activity. Zinc inhibition fit the competitive inhibitor model. The inhibitory effect of zinc occurred only with citrate as substrate and was specific for the citrate --> cis-aconitate reaction. Other cations (Ca2+, Mn2+, Cd2+) did not result in the inhibitory effects obtained with zinc. The presence of endogenous zinc inhibited the m-aconitase activity of the prostate mitochondrial preparations. Kidney preparations that contain lower endogenous zinc levels exhibited no endogenous inhibition of m-aconitase activity. Studies with pig prostate and seminal vesicle mitochondrial preparations also revealed that zinc was a competitive inhibitor against citrate of m-aconitase activity. The effects of zinc on purified beef heart m-aconitase verified the competitive inhibitor action of zinc. In contrast, zinc had no inhibitory effect on purified cytosolic aconitase. These studies reveal for the first time that zinc is a specific inhibitor of m-aconitase of mammalian cells. In prostate epithelial cells, in situ mitochondrial zinc levels inhibit m-aconitase activity, which provides a mechanism by which citrate oxidation is limited.