NADPH-dependent generation of a cytosolic dithiol which activates hepatic iodothyronine 5′-deiodinase. Demonstration by alkylation with iodoacetamide

Das, Arun K.; Hummel, Brian C. W.; Walfish, Paul G.

doi:10.1042/bj2400559

Cited by 8 publications

(9 citation statements)

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“…Three components of CCS, DFA, DFB and NADPH, regulate the activation of 5'-DI by this CCS [7], and previous studies [7,8] are consistent with the proposal that the degree of activation by this system depends on the concentration of reduced DFB available for the reduction of the oxidized enzyme, ESI [19,20]. In the present study, the experimental conditions in which the concentration of reduced DFB depends on the amount of oxidized DFB were manipulated by varying the concentration of DFB at fixed concentrations of DFA and a constant excess of NADPH [8]. Since preliminary studies with nanomolar concentrations of rT3as substrate showed the optimal pH to be 7.5 and the optimal temperature to be 37°C for the reaction with both liver and kidney preparations, all experiments, whether performed with partially or highly purified CCS components, used these assay conditions.…”

Section: Purification Of Dfa and Dfbmentioning

confidence: 61%

“…Evidence has been presented suggesting that this system contains a cytosolic dithiol component of Mr approx. 13 000 (DFB) that is reduced by another cytosolic component of Mr > 60000 (DFA) (a putative reductase) in the presence of NADPH [7,8]. These studies utilized relatively crude cytosolic Fractions A and B containing DFA and DFB respectively and showed that this endogenous cofactor system, which supported the activity of a type I low-Km 5'-DI, had properties similar to those of the thioredoxin system [7][8][9].…”

Section: Introductionmentioning

confidence: 99%

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Kinetic characteristics of a thioredoxin-activated rat hepatic and renal low-Km iodothyronine 5′-deiodinase

Bhat

Iwase

Hummel

et al. 1989

Biochemical Journal

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The properties and kinetic characteristics of a non-GSH NADPH-dependent cofactor system activating rat hepatic and renal 5'-deiodinase (5'-DI), which we have previously demonstrated with partially purified cytosol Fractions A and B [Sawada, Hummel & Walfish (1986) Biochem. J. 234, 391-398], were examined further. Although microsomal fractions prepared from either rat liver or kidneys could be activated by crude cytosol Fractions A and B from those tissues as well as from rat brain and heart, a homologous hepatic or renal system was the most potent in producing 5'-deiodination of reverse tri-iodothyronine (rT3). At nanomolar concentrations both rT3 and thyroxine (T4) were deiodinated but with a much greater substrate preference for rT3 than for T4. However, at micromolar concentrations of these substrates no activation of 5'-DI could be detected. In this deiodinative system, T4 and tri-iodothyronine (T3) competitively inhibited 5'-deiodination of rT3. Dicoumarol, iopanoate, arsenite and diamide were also inhibitory to the activation of hepatic or renal 5'-deiodination by this cofactor system. Purification of cofactor components in hepatic crude cytosolic Fractions A and B to near homogeneity, as assessed by their enzymic and physical properties, indicated that these co-purified with and were therefore identical with thioredoxin reductase and thioredoxin respectively, and accounted almost entirely for the observed activation of rT3 5'-DI. When highly purified liver cytosolic thioredoxin reductase and thioredoxin were utilized to determine the kinetic characteristics of the reaction, evidence for a sequential mechanism operative at nanomolar but not micromolar concentrations of rT3 and T4 was obtained. The Km for rT3 was 1.4 nM. Inhibition by 6-n-propyl-2-thiouracil (Ki 6.7 microM) was competitive with respect to thioredoxin and non-competitive with respect to rT3, whereas inhibition by T4 (Ki 1.3 microM) was competitive. Since rT3 is a potent inhibitor of T4 5'-deiodination, this thioredoxin system activating deiodination of rT3 may play an important role in regulating the rate of intracellular production of T3 from T4.

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Section: Purification Of Dfa and Dfbmentioning

confidence: 61%

Section: Introductionmentioning

confidence: 99%

Kinetic characteristics of a thioredoxin-activated rat hepatic and renal low-Km iodothyronine 5′-deiodinase

Bhat

Iwase

Hummel

et al. 1989

Biochemical Journal

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“…In vitro deiodinase assays usually use the nonphysiological reducing agent DTT, but Goswami and Rosenberg (21) purified from cells a protein thiol cofactor resembling glutaredoxin (Grx). Another group identified Trx and Trx reductase (TrxR) as cytosolic factors needed for Dio1 reduction (22)(23)(24), consistent with decreased Dio1 activity under conditions decreasing reduced Trx (25). Dio1 activity from liver measured with a physiological NADPH-dependent Trx/TrxR regenerating system was lower than with millimolar DTT but revealed a K m (Trx) of 1.8 μM and a K m [reverse T 3 (rT 3 )] of 1.4 nM, as well as sequential kinetics as in Dio2 and Dio3 (24).…”

mentioning

confidence: 99%

Crystal structure of mammalian selenocysteine-dependent iodothyronine deiodinase suggests a peroxiredoxin-like catalytic mechanism

Schweizer

Schlicker

Braun

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

161

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Local levels of active thyroid hormone (3,3′,5-triiodothyronine) are controlled by the action of activating and inactivating iodothyronine deiodinase enzymes. Deiodinases are selenocysteine-dependent membrane proteins catalyzing the reductive elimination of iodide from iodothyronines through a poorly understood mechanism. We solved the crystal structure of the catalytic domain of mouse deiodinase 3 (Dio3), which reveals a close structural similarity to atypical 2-Cys peroxiredoxin(s) (Prx). The structure suggests a route for proton transfer to the substrate during deiodination and a Prx-related mechanism for subsequent recycling of the transiently oxidized enzyme. The proposed mechanism is supported by biochemical experiments and is consistent with the effects of mutations of conserved amino acids on Dio3 activity. Thioredoxin and glutaredoxin reduce the oxidized Dio3 at physiological concentrations, and dimerization appears to activate the enzyme by displacing an autoinhibitory loop from the iodothyronine binding site. Deiodinases apparently evolved from the ubiquitous Prx scaffold, and their structure and catalytic mechanism reconcile a plethora of partly conflicting data reported for these enzymes.hyroid hormones regulate mammalian development as well as energy expenditure and metabolism in the adult (1). The active, nuclear receptor-binding form of thyroid hormone is 3,3′,5-triiodothyronine (T 3 ). The thyroid gland mainly releases thyroxine [3,3′,5,5′-tetraiodothyronine (T 4 ); Fig. 1A], and T 3 is formed and degraded through elimination of iodine atoms from the 5′-and 5-positions, respectively (2) (Fig. S1). Three types of deiodinase enzymes are involved in activation and inactivation of thyroid hormones (Fig. S1). They form a family of transmembrane enzymes with homologous catalytic domains (2). Type II deiodinase (Dio2) catalyzes the activating (outer ring) 5′-deiodination of the prohormone T 4 to T 3 , whereas type III deiodinase (Dio3) catalyzes inactivating (inner ring) 5-deiodination (2). Type I deiodinase (Dio1) is capable of both types of reactions (3). Cells targeted by the hormone can thereby fine-tune intracellular T 3 levels through deiodinase expression according to their needs during development or upon metabolic challenges. Dio1, Dio2, and Dio3 share a conserved amino acid sequence and a selenocysteine residue essential for efficient deiodination, although nonmammalian cysteine-deiodinases have been found (4, 5). Although most of the characterized selenoproteins act as peroxidases or protein reductases, deiodinases are the only selenoenzymes known to catalyze halogen eliminations from aromatic rings. Despite a large body of experimental data, there is no coherent model for deiodinase catalysis that incorporates the bulk of available data. Results and DiscussionTo explore the unique deiodinase catalytic mechanism, we determined the crystal structure of the cytoplasmic catalytic domain of Mus musculus Dio3 (Dio3 cat ) with Sec 170 replaced by cysteine ( Fig. 1 B and C and Table 1). The structu...

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“…NOTE ADDED: After completion of the studies described above, our earlier suggestion that the thioredoxin system could account for the observed properties of the postulated DFA -DFB -NADPH 5'-deiodinase cofactor system, which operates at nanomolar but not micromolar concentrations of rT3 (Sawada et al 1986a;Das et al.…”

Section: Resultsmentioning

confidence: 94%

“…Although vitro 5'-deiodination of iodothyronines by isolated loss of cytosolic stimulating activity during starvation hepatic microsomes is markedly dependent upon added and its panial restoration GSH and NADPH thiols (Visser et al 1976; Goswami and Rosenberg and Ingbar 1979;Harris etal. 1979) suggest that GSH is the principal endogenous cofactor for deiodinase, other findings (Gavin et al 1980 (DFB) of Mr = 13 000 in fraction B and NADPH 'Author to whom all correspondence should be addressed at (Sawada et al 1986a(Sawada et al , 1986bDas et al 1986 For personal use only. (Holmgren 1985).…”

Section: Introductionmentioning

confidence: 97%

Bacterial and mammalian thioredoxin systems activate iodothyronine 5′-deiodination

Das

Hummel

Gleason

et al. 1988

Biochem. Cell Biol.

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The identity of a dithiol (designated DFB) of relative mass (Mr) = 13,000, reported previously to be present in fraction B of rat liver cytosol and to participate as a cofactor in the 5'-deiodination of iodothyronines, has been investigated. Substitution of highly purified thioredoxin from Escherichia coli for fraction B or of highly purified thioredoxin reductase from either E. coli or rat liver for cytosolic fraction A (containing DFB reductase) permits deiodination of 3,3',5'-[125I]triiodothyronine by rat liver microsomes to proceed. Addition of antibodies to highly purified rat-liver thioredoxin or thioredoxin reductase inhibits deiodination. Thus, the thioredoxin system largely accounts for the activity of the cytosolic cofactor system supporting 5'-deiodination of 3,3',5'-triiodothyronine in rat liver.

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NADPH-dependent generation of a cytosolic dithiol which activates hepatic iodothyronine 5′-deiodinase. Demonstration by alkylation with iodoacetamide

Cited by 8 publications

References 25 publications

Kinetic characteristics of a thioredoxin-activated rat hepatic and renal low-Km iodothyronine 5′-deiodinase

Kinetic characteristics of a thioredoxin-activated rat hepatic and renal low-Km iodothyronine 5′-deiodinase

Crystal structure of mammalian selenocysteine-dependent iodothyronine deiodinase suggests a peroxiredoxin-like catalytic mechanism

Bacterial and mammalian thioredoxin systems activate iodothyronine 5′-deiodination

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