Species differences in liver type I iodothyronine deiodinase.

The type I iodothyronine deiodinase (ID-I) of liver is an important enzyme for the conversion of the prohormone thyroxine (T4) to the active thyroid hormone 3,3',5-triiodothyronine (T3). Because it is an integral membrane protein of low abundance, purification of ID-I from rat liver has proven to be difficult. We have analyzed ID-I in liver microsomal fractions from various animals to reveal possible species differences and to explore alternative sources for the isolation of the enzyme. ID-I was characterized by enzyme assay with 3,3',5'-triiodothyronine (rT3) as the preferred substrate and by affinity-labeling with N-bromoacetyl-[125I]T3 (BrAc[125I]T3). Labeled ID-I subunit was identified and quantified by SDS-PAGE and autoradiography. The Mr of ID-I in the species investigated varied between 25.7 and 29.1 kDa. Rat and dog liver microsomes had a markedly higher enzyme content than microsomes of human, mouse, rabbit, cow, pig, sheep, goat, chicken or duck liver. Rat liver microsomes showed the highest ID-I activity of all species examined. Turnover numbers for ID-I varied between 264 and 1059 min-1, with rabbit and goat showing the highest values. However, dog liver ID-I displayed an exceptionally low turnover number of 78 min-1. In conclusion, ID-I has similar properties in all species examined with the notable exception of dog.     

Identification of type I iodothyronine 5'-deiodinase as a selenoenzyme

A 27.8 kDa membrane selenoprotein was previously identified in rat thyroid, liver and kidney, the tissues with the highest activities of type I iodothyronine 5'-deiodinase. This membrane enzyme catalyzes the deiodination of L-thyroxine to the biologically active thyroid hormone 3,3',5-triiodothyronine. A decrease in the activity of this enzyme, observed here in the liver of selenium-deficient rats, was found to be due to the absence of a selenium-dependent membrane-bound component. By chemical and enzymatic fragmentation of the 75Se-labeled selenoprotein and of the 27 kDa substrate binding type I 5'-deiodinase subunit, affinity-labeled with N-bromoacetyl-[125I]L-thyroxine, and comparison of the tracer distribution in the peptide fragments the identity of the two proteins was shown. The data indicate that the deiodinase subunit contains one selenium atom per molecule and suggest that a highly reactive selenocysteine is the residue essential for the catalysis of 5'-deiodination. From the results it can be concluded that type I iodothyronine 5'-deiodinase is a selenoenzyme.     

Selenocysteine confers the biochemical properties characteristic of the type I iodothyronine deiodinase.

The conversion of thyroxine to 3,5,3'-triiodothyronine (T3) is the first step in thyroid hormone action, and the Type I iodothyronine deiodinase supplies most of this extrathyroidal T3 in the rat. We found that the cDNA coding for this enzyme contains an in-frame UGA encoding the rare amino acid selenocysteine. Using site-directed mutagenesis, we have converted selenocysteine to cysteine and expressed the wild-type and cysteine mutant enzymes in JEG-3 cells by transient transfection. The kinetic properties of the transiently expressed wild-type enzyme are nearly identical to those reported for rat liver Type I deiodinase. Substitution of sulfur for selenium causes a 10-fold increase in the Km of the enzyme for the favored substrate 3,3',5'-triiodothyronine (rT3), a 100-fold decrease in the sensitivity of rT3 deiodination to competitive inhibition by gold and a 300-fold increase in the apparent Ki for uncompetitive inhibition by 6-n-propylthiouracil. These results demonstrate that selenium is responsible for the biochemical properties which characterize Type I iodothyronine monodeiodination.     

Identification of essential histidine residues in rat type I iodothyronine deiodinase.

Deiodination is required for conversion of thyroxine, the inactive prohormone secreted by the thyroid gland, to 3,5,3'-triiodothyronine, the biologically active thyroid hormone. The principal enzyme catalyzing this reaction, Type I iodothyronine 5' deiodinase, was shown recently to contain the amino acid, selenocysteine, and site-directed mutagenesis showed that this amino acid confers the biochemical properties characteristic of this enzyme. Previous studies suggest that a histidine residue may also be critical for activity. To further our understanding of the biochemical mechanism of this reaction, we have used in vitro mutagenesis to examine the contribution of each of the 4 histidines in this enzyme to the deiodination process. Two of the histidines (185 and 253) are not involved in deiodination, as their removal had no effect on activity. Mutagenesis of histidine 158 resulted in complete loss of activity, suggesting a role in either protein conformation or catalysis. The most informative results were obtained from the studies of histidine 174. Mutagenesis of this histidine to asparagine or glutamine altered reactivity with substrate and reduced inhibition by diethylpyrocarbonate and rose bengal. These results demonstrate that histidine 174 is critical to function and appears to be involved in binding of hormone.     

Characterization of the subunit structure of the catalytically active type I iodothyronine deiodinase

Type I iodothyronine deiodinase is a approximately 50-kDa, integral membrane protein that catalyzes the outer ring deiodination of thyroxine. Despite the identification and cloning of a 27-kDa selenoprotein with the catalytic properties of the type I enzyme, the composition and the physical nature of the active deiodinase are unknown. In this report, we use a molecular approach to determine holoenzyme composition, the role of the membrane anchor on enzyme assembly, and the contribution of individual 27-kDa subunits to catalysis. Overexpression of an immunologically unique rat 27-kDa protein in LLC-PK1 cells that contain abundant catalytically active 27-kDa selenoprotein decreased deiodination by approximately 50%, and > 95% of the LLC-PK1 derived 27-kDa selenoprotein was specifically immune precipitated by the anti-rat enzyme antibody. The hybrid enzyme had a molecular mass of 54 kDa and an s(20,w) of approximately 3.5 S indicating that every native 27-kDa selenoprotein partnered with an inert rat 27-kDa subunit in a homodimer. Enzyme assembly did not depend on the presence of the N-terminal membrane anchor of the 27-kDa subunit. Direct visualization of the deiodinase dimer showed that the holoenzyme was sorted to the basolateral plasma membrane of the renal epithelial cell.     

Rat liver type I iodothyronine deiodinase is not identical to protein disulfide isomerase

This study was done to test the recent hypothesis (Boado et al. (1988) Biochem. Biophys. Res. Commun. 155, 1297-1304) that type I iodothyronine deiodinase (ID-I) is identical to protein disulfide isomerase (PDI). Autoradiograms of rat liver microsomal proteins, labeled with N-bromoacetyl-[125I]triiodothyronine (BrAc[125I]T3) and separated by SDS-PAGE, show predominantly 2 radioactive bands of Mr 27 and 56 kDa. Substrates and inhibitors of ID-I inhibited labeling of the 27 kDa band but not that of the 56 kDa band. Treatment of microsomes with trypsin abolished labeling of the 27 kDa protein and destroyed the activity of ID-I but did not prevent labeling of the 56 kDa protein. Following treatment of microsomes at pH 8.0-9.5 or with 0.05% deoxycholate (DOC) PDI content and labeling of the 56 kDa protein were strongly diminished but ID-I activity and labeling of the 27 kDa protein were not affected. The latter decreased in parallel after treatment at pH greater than or equal to 10. Rat pancreas microsomes contain high amounts of PDI but show no ID-I activity. Reaction of these microsomes with BrAc[125I]T3 results in extensive labeling of a 56 kDa protein but no labeling of a 27 kDa protein. Pure PDI (Mr 56 kDa) was readily labeled by BrAc[125I]T3 but showed no deiodinase activity. These results strongly suggest that the 27 kDa band represents (a subunit of) ID-I while the 56 kDa band represents PDI. From these and other data it is concluded that PDI and ID-I are not identical proteins.     

Inhibition of rat liver iodothyronine deiodinase. Interaction of aurones with the iodothyronine ligand-binding site

We report that aurone derivatives of plant extracts produce potent, dose-dependent, and ultimately complete inhibition of three different metabolic monodeiodination pathways catalyzed by rat liver microsomal type I iodothyronine deiodinase. These data show that (3'),4',4,6-(tetra)trihydroxyaurones are the most potent naturally occurring plant-derived inhibitors of this deiodinase enzyme (IC50 V 0.5 microM). Lineweaver-Burk analysis using both L-thyroxine (T4) and 3',5',3-triiodothyronine as substrates suggests a cofactor competitive mechanism of inhibition for 4',4,6-trihydroxyaurone which also can displace 125I-L-T4 from binding to thyroxine-binding prealbumin with a potency comparable to its inhibition of T4-5'-deiodinase. Among type I deiodinase inhibitors, cofactor competition has been observed only for propylthiourea. Computer graphic modeling studies were also carried out to explore aurone conformations and to compare them with those of the thyroid hormones. This analysis shows that the aurones can adopt either a planar or an antiskewed conformation, such as observed for 3',5',3-triiodothyronine, the most potent natural deiodinase substrate inhibitor. The thyroxine-binding prealbumin complex was used to model the deiodinase ligand binding site because of the similarity observed between inhibitor binding affinity and enzyme inhibition characteristics. These studies show that the aurones which adopt an antiskewed conformation can interact favorably in the prealbumin binding site. This model of the deiodinase active site can be used to design other deiodinase inhibitors.     

Inhibition of type I and type II iodothyronine deiodinase activity in rat liver, kidney and brain produced by selenium deficiency.

Selenium deficiency for periods of 5 or 6 weeks in rats produced an inhibition of tri-iodothyronine (T3) production from added thyroxine (T4) in brain, liver and kidney homogenate. This inhibition was reflected in plasma T4 and T3 concentrations, which were respectively increased and decreased in selenium-deficient animals. Although plasma T4 levels increased in selenium-deficient animals, this did not produce the normal feedback inhibition on thyrotropin release from the pituitary. Selenium deficiency was confirmed in the animals by decreased selenium-dependent glutathione peroxidase (Se-GSH-Px) activity in all of these tissues. Administration of selenium, as a single intraperitoneal injection of 200 micrograms of selenium (as Na2SeO3)/kg body weight completely reversed the effects of selenium deficiency on thyroid-hormone metabolism and partly restored the activity of Se-GSH-Px. Selenium administration at 10 micrograms/kg body weight had no significant effect on thyroid-hormone metabolism or on Se-GSH-Px activity in any of the tissues studied. The characteristic changes in plasma thyroid-hormone levels that occurred in selenium deficiency appeared not to be due to non-specific stress factors, since food restriction to 75% of normal intake or vitamin E deficiency produced no significant changes in plasma T4 or T3 concentration. These data are consistent with the view that the Type I and Type II iodothyronine deiodinase enzymes are seleno-enzymes or require selenium-containing cofactors for activity.     

Substituted 2-oxo-azepane derivatives are potent, orally active gamma-secretase inhibitors

A hydroxamic acid screening hit 1 was elaborated to 5,5-dimethyl-2-oxoazepane derivatives exhibiting low nanomolar inhibition of gamma-secretase, a key proteolytic enzyme involved in Alzheimer's disease. Early ADME data showed a high metabolic clearance for the geminal dimethyl analogs which could be overcome by replacement with the bioisosteric geminal difluoro group. Synthesis and structure-activity relationship are discussed and in vivo active compounds are presented.     

Gamma-lactone-Functionalized antitumoral acetogenins are the most potent inhibitors of mitochondrial complex I

To study the relevance of the terminal alpha,beta-unsaturated gamma-methyl-gamma-lactone moiety of the antitumoral acetogenins of Annonaceae for potent mitochondrial complex I inhibition, we have prepared a series of semisynthetic acetogenins with modifications only in this part of the molecule, from the natural rolliniastatin-1 (1) and cherimolin-1 (2). Some of the hydroxylated derivatives (1b, 1d and 1e) in addition to two infrequent natural beta-hydroxy gamma-methyl gamma-lactone acetogenins, laherradurin (3) and itrabin (4), are more potent complex I inhibitors than any other known compounds.     

The human type 2 iodothyronine deiodinase is a selenoprotein highly expressed in a mesothelioma cell line.

Types 1 and 3 iodothyronine deiodinases are known to be selenocysteine-containing enzymes. Although a putative human type 2 iodothyronine deiodinase (D2) gene (hDio2) encoding a similar selenoprotein has been identified, basal D2 activity is not selenium (Se)-dependent nor has D2 been labeled with (75)Se. A human mesothelioma cell line (MSTO-211H) has recently been shown to have approximately 40-fold higher levels of hDio2 mRNA than mesothelial cells. Mesothelioma cell lysates activate thyroxine (T(4)) to 3,5,3'-triiodothyronine with typical characteristics of D2 such as low K(m) (T(4)), 1.3 nm, resistance to propylthiouracil, and a short half-life ( approximately 30 min). D2 activity is approximately 30-fold higher in Se-supplemented than in Se-depleted medium. An antiserum prepared against a peptide deduced from the Dio2 mRNA sequence precipitates a (75)Se protein of the predicted 31-kDa size from (75)Se-labeled mesothelioma cells. Bromoadenosine 3'5' cyclic monophosphate increases D2 activity and (75)Se-p31 approximately 2.5-fold whereas substrate (T(4)) reduces both D2 activity and (75)Se-p31 approximately 2-3-fold. MG132 or lactacystin (10 microm), inhibitors of the proteasome pathway by which D2 is degraded, increase both D2 activity and (75)Se-p31 3-4-fold and prevent the loss of D2 activity during cycloheximide or substrate (T(4)) exposure. Immunocytochemical studies with affinity-purified anti-hD2 antibody show a Se-dependent increase in immunofluorescence. Thus, human D2 is encoded by hDio2 and is a member of the selenodeiodinase family accounting for its highly catalytic efficiency in T(4) activation.     

Polyarginines are potent furin inhibitors

The ubiquitous serine endoprotease furin has been implicated in the activation of bacterial toxins and viral glycoproteins as well as in the metastatic progression of certain tumors. Although high molecular mass bioengineered serpin inhibitors have been well characterized, no small nontoxic nanomolar inhibitors have been reported to date. Here we describe the identification of such inhibitors using positional scanning amidated and acetylated synthetic l- and d-hexapeptide combinatorial libraries. The results indicated that l-Arg or l-Lys in all positions generated the most potent inhibitors. However, further investigation revealed that the peptide terminating groups hindered inhibition. Consequently, a series of non-amidated and acetylated polyarginines was synthesized. The most potent inhibitor identified, nona-l-arginine, had a K(i) for furin of 40 nm. The K(i) values for the related convertases PACE4 and prohormone convertase-1 (PC1) were 110 nm and 2.5 microm, respectively. Although nona-l-arginine was cleaved by furin, the major products after a 6-h incubation at 37 degrees C were hexa- and hepta-l-arginines, both of which retained the great majority of their potency and specificity against furin. Hexa-d-arginine was as potent and specific a furin inhibitor as hexa-l-arginine (K(i) values of hexa-d-arginine: 106 nm, 580 nm, and 13.2 microm for furin, PACE4, and PC1, respectively). PC2 was not inhibited by any polyarginine tested; indeed, PC2 showed an increase in activity of up to 140% of the control in the presence of l-polyarginines. Data are also presented that show extended subsite recognition by furin and PC2. Whereas N-terminal acetylation was found to reduce the inhibitory potency of the l-hexapeptide LLRVKR against furin 8-fold, C-terminal amidation reduced the potency < 2-fold. Conversely, N-terminal acetylation increased the potency against PC2 nearly 3-fold, whereas C-terminal amidation of the same peptide increased the potency by a factor of 1.6. Our data indicate that non-acetylated, poly-d-arginine-derived molecules may represent excellent lead compounds for the development of therapeutically useful furin inhibitors.     

5-Substituted 3-chlorokenpaullone derivatives are potent inhibitors of Trypanosoma brucei bloodstream forms

Trypanothione synthetase is an essential enzyme for kinetoplastid parasites which cause highly disabling and fatal diseases in humans and animals. Inspired by the observation that N(5)-substituted paullones inhibit the trypanothione synthetase from the related parasite Leishmania infantum, we designed and synthesized a series of new derivatives. Although none of the new compounds displayed strong inhibition of Trypanosoma brucei trypanothione synthetase, several of them caused a remarkable growth inhibition of cultivated Trypanosoma brucei bloodstream forms. The most potent congener 3a showed antitrypanosomal activity in double digit nanomolar concentrations and a selectivity index of three orders of magnitude versus murine macrophage cells.     

Discovery of highly potent and selective type I B-Raf kinase inhibitors

A series of pyrazolo[1,5-α]pyrimidine analogs has been prepared and found to be potent and selective B-Raf inhibitors. Molecular modeling suggests they bind to the active conformation of the enzyme.     

Sex steroids modulate rat anterior pituitary and liver iodothyronine deiodinase activities.

In this study, we investigated the sex hormone regulation of 5'-iodothyronine deiodinase activity, which is responsible for enzymatic conversion of thyroxine into the bioactive form, triiodothyronine. Pituitary homogenates and liver microsomes from: 1) ovariectomized rats injected with 17-beta-estradiol benzoate and/or progesterone (0.7 and 250 microg/100 g body weight, respectively, subcutaneously, over 10 days); 2) male castrated rats treated or not with 0.4 mg/100 g body weight testosterone propionate, intramuscular, over 7 days, were assayed for type 1 and type 2 deiodinase activity in the pituitary. Enzyme activities were measured by release of (125)I from deiodination of (125)I reverse triiodothyronine under varying assay conditions. Estrogen stimulated anterior pituitary and liver type 1 deiodinase activity in ovariectomized rats (45 and 30 %, p < 0.05). Progesterone inhibited the liver enzyme (40 %, p < 0.05), and had no effect on the pituitary, but in both tissues, blocked estrogen stimulatory effect on type 1 deiodinase. In males, testosterone normalized the reduced liver type 1 deiodinase of castrated rats. However, in the pituitary, castration increased (50 %) type 1 deiodinase independent of testosterone treatment, suggesting the existence of a inhibitory testicular regulator of pituitary type 1 enzyme. Treatments did not alter pituitary type 2 deiodinase activity. In conclusion, gonads and sex steroids differentially modulate type 1 deiodinase activity in rat pituitary and liver.