309 research outputs found
Thyroid hormone deiodination
The enzymatic deiodination of thyroid hormone is an important process
since it concerns- among other things- the regulation of thyromimetic activity
at the site of the target organ. To understand the mechanism of this
regulation it is necessary to have a detailed knowledge of the mode of action
of the enzyme(s) involved in the metabolism of thyroid hormone. My investigations
of the deiodination of iodothyronines at the subcellular level, forming
the basis of this thesis, are described in the appendix papers. It is not
intended to deal in extenso with the technical aspects of my studies in the
preceeding chapters. Rather it will be attempted to give a general review of
the literature including- with some emphasis -my own work.
Though not directly related to the subject of this thesis, the biosynthesis
of thyroid hormone in the thyroid gland is treated in the first
chapter. This is done because of possible similarities between thyroid hormone
iodination and deiodination pathways, which are suggested by the finding that
some drugs inhibit both processes. In the same chapter the relationship
between iodothyronine structure and biological potency is described to illustrate
that indeed deiodination has a dramatic effect on the activity of
thyroid hormone. Besides deiodination, other pathways of metabolism are also
considered.
The second chapter concerns the in vivo investigation of thyroid hormone
deiodination under physiological and pathological conditions. This includes
the effects of internal and external factors which affect deiodination, such
as dietary intake, drugs, stress and illness. Since much work has been done
to find an explanation for the effect of calorie restriction on deiodination
at the molecular level, the role of the diet is emphasized. This appears
particularly important since nutritional status must be considered to contribute
to the change in thyroid hormone metabolism found in other situations,
for example in systemic illness.
The in vitro observations of the enzymatic deiodination of thyroid
hormone are described in chapter 3. A distinction has been made between
(early) reports on the analysis of iodide production using chromatography,
and (more recent) studies dealing with the detection of specific metabolites,
often by means of radioimmunoassay. My investigations which belong to the
latter category are presented in the appendix paper
Substitution of cysteine for selenocysteine in the catalytic center of type III iodothyronine deiodinase reduces catalytic efficiency and alters substrate preference
Human type III iodothyronine deiodinase (D3) catalyzes the conversion of
T(4) to rT(3) and of T(3) to 3, 3'-diiodothyronine (T2) by inner-ring
deiodination. Like types I and II iodothyronine deiodinases, D3 protein
contains selenocysteine (SeC) in the highly conserved core catalytic
center at amino acid position 144. To evaluate the contribution of SeC144
to the catalytic properties of D3 enzyme, we generated mutants in which
cysteine (D3Cys) or alanine (D3Ala) replaces SeC144 (D3wt). COS cells were
transfected with expression vectors encoding D3wt, D3Cys, or D3Ala
protein. Kinetic analysis was performed on homogenates with dithiothreitol
as reducing cofactor. The Michaelis constant of T(3) was 5-fold higher for
D3Cys than for D3wt protein. In contrast, the Michaelis constant of T(4)
increased 100-fold. The D3Ala protein was enzymatically inactive.
Semiquantitative immunoblotting of homogenates with a D3 antiserum
revealed that about 50-fold higher amounts of D3Cys and D3Ala protein are
expressed relative to D3wt protein. The relative substrate turnover number
of D3Cys is 2-fold reduced for T(3) and 6-fold reduced for T(4)
deiodination, compared with D3wt enzyme. Studies in intact COS cells
expressing D3wt or D3Cys showed that the D3Cys enzyme is also active under
in situ conditions. In conclusion, the SeC residue in the catalytic center
of D3 is essential for efficient inner-ring deiodination of T(3) and in
particular T(4) at physiological substrate concentrations
Substitution of cysteine for a conserved alanine residue in the catalytic center of type II iodothyronine deiodinase alters interaction with reducing cofactor
Human type II iodothyronine deiodinase (D2) catalyzes the activation of
T(4) to T(3). The D2 enzyme, like the type I (D1) and type III (D3)
deiodinases, contains a selenocysteine (SeC) residue (residue 133 in D2)
in the highly conserved catalytic center. Remarkably, all of the D2
proteins cloned so far have an alanine two residue-amino terminal to the
SeC, whereas all D1 and D3 proteins contain a cysteine at this position. A
cysteine residue in the catalytic center could assist in enzymatic action
by providing a nucleophilic sulfide or by participating in redox reactions
with a cofactor or enzyme residues. We have investigated whether D2
mutants with a cysteine (A131C) or serine (A131S) two-residue amino
terminal to the SeC are enzymatically active and have characterized these
mutants with regard to substrate affinity, reducing cofactor interaction
and inhibitor profile. COS cells were transfected with expression vectors
encoding wild-type (wt) D2, D2 A131C, or D2 A131S proteins. Kinetic
analysis was performed on homogenates with dithiothreitol (DTT) as
reducing cofactor. The D2 A131C and A131S mutants displayed similar
Michaelis-Menten constant values for T(4) (5 nM) and reverse T(3) (9 nM)
as the wt D2 enzyme. The limiting Michaelis-Menten constant for DTT of the
D2 A131C enzyme was 3-fold lower than that of the wt D2 enzyme. The wt and
mutant D2 enzymes are essentially insensitive to propylthiouracil
[concentration inhibiting 50% of activity (IC(50)) > 2 mM] in the presence
of 20 mM DTT, but when tested in the presence of 0.2 mM DTT the IC(50)
value for propylthiouracil is reduced to about 0.1 mM. During incubations
of intact COS cells expressing wt D2, D2 A131C, or D2 A131S, addition of
increasing amounts of unlabeled T(4) resulted in the saturation of
[(125)I]T(4) deiodination, as reflected in a decrease of [(125)I]T(3)
release into the medium. Saturation first appeared at medium T(4)
concentrations between 1 and 10 nM. In conclusion: substitution of
cysteine for a conserved alanine residue in the catalytic center of the D2
protein does not inactivate the enzyme in vitro and in situ, but rather
improves the interaction with the reducing cofactor DTT in vitro
Identification of markers associated with bacterial blight resistance loci in cowpea (Vigna unguiculata (L.) Walp.)
Cowpea bacterial blight (CoBB), caused by Xanthomonas axonopodis pv. vignicola (Xav), is a worldwide major disease of cowpea [Vigna unguiculata (L.) Walp.]. Among different strategies to control the disease including cultural practices, intercropping, application of chemicals, and sowing pathogen-free seeds, planting of cowpea genotypes with resistance to the pathogen would be the most attractive option to the resource poor cowpea farmers in sub-Saharan Africa. Breeding resistance cultivars would be facilitated by marker-assisted selection (MAS). In order to identify loci with effects on resistance to this pathogen and map QTLs controlling resistance to CoBB, eleven cowpea genotypes were screened for resistance to bacterial blight using 2 virulent Xav18 and Xav19 strains isolated from Kano (Nigeria). Two cowpea genotypes Danila and Tvu7778 were identified to contrast in their responses to foliar disease expression following leaf infection with pathogen. A set of recombinant inbred lines (RILs) comprising 113 individuals derived from Danila (resistant parent) and Tvu7778 (susceptible parent) were infected with CoBB using leaf inoculation method. The experiments were conducted under greenhouse conditions (2007 and 2008) and disease severity was visually assessed using a scale where 0 = no disease and 4 = maximum susceptibility with leaf drop. A single nucleotide polymorphism (SNP) genetic map with 282 SNP markers constructed from the same RIL population was used to perform QTL analysis. Using Kruskall-Wallis and Multiple-QTL model of MapQTL 5, three QTLs, CoBB-1, CoBB-2 and CoBB-3 were identified on linkage group LG3, LG5 and LG9 respectively showing that potential resistance candidate genes cosegregated with CoBB resistance phenotypes. Two of the QTLs CoBB-1, CoBB-2 were consistently confirmed in the two experiments accounting for up to 22.1 and to 17.4% respectively for the first and second experiments. Whereas CoBB-3 was only discovered for the first experiment (2007) with less phenotypic variation explained of about 10%. Our results represent a resource for molecular marker development that can be used for marker assisted selection of bacterial blight resistance in cowpe
Further insights into the allan-herndon-dudley syndrome: Clinical and functional characterization of a novel MCT8 mutation
Background. Mutations in the thyroid hormone (TH) transporter MCT8 have been identified as the cause for Allan-Herndon-Dudley Syndrome (AHDS), characterized by severe psychomotor retardation and altered TH serum levels. Here we report a novel MCT8 mutation identified in 4 generations of one family, and its functional characterization. Methods. Proband and family members were screened for 60 genes involved in X-linked cognitive impairment and the MCT8 mutation was confirmed. Functional consequences of MCT8 mutations were studied by analysis of [125I]TH transport in fibroblasts and transiently transfected JEG3 and COS1 cells, and by subcellular localization of the transporter. Results. The proband and a male cousin demonstrated clinical findings characteristic of AHDS. Serum analysis showed high T3, low rT3, and normal T4 and TSH levels in the proband. A MCT8 mutation (c.869C>T; p.S290F) was identified in the proband, his cousin, and several female carriers. Functional analysis of the S290F mutant showed decreased TH transport, metabolism and protein expression in the three cell types, whereas the S290A mutation had no effect. Interestingly, both uptake and efflux of T3 and T4 was impaired in fibroblasts of the proband, compared to his healthy brother. However, no effect of the S290F mutation was observed on TH efflux from COS1 and JEG3 cells. Immunocytochemistry showed plasma membrane localization of wild-type MCT8 and the S290A and S290F mutants in JEG3 cells. Conclusions. We describe a novel MCT8 mutation (S290F) in 4 generations of a family with Allan-Herndon-Dudley Syndrome. Functional analysis demonstrates loss-of-function of the MCT8 transporter. Furthermore, our results indicate that the function of the S290F mutant is dependent on cell context. Comparison of the S290F and S290A mutants indicates that it is not the loss of Ser but its substitution with Phe, which leads to S290F dysfunction
Characterization of iodothyronine sulfatase activities in human and rat liver and placenta
In conditions associated with high serum iodothyronine sulfate
concentrations, e.g. during fetal development, desulfation of these
conjugates may be important in the regulation of thyroid hormone
homeostasis. However, little is known about which sulfatases are involved
in this process. Therefore, we investigated the hydrolysis of
iodothyronine sulfates by homogenates of V79 cells expressing the human
arylsulfatases A (ARSA), B (ARSB), or C (ARSC; steroid sulfatase), as well
as tissue fractions of human and rat liver and placenta. We found that
only the microsomal fraction from liver and placenta hydrolyzed
iodothyronine sulfates. Among the recombinant enzymes only the endoplasmic
reticulum-associated ARSC showed activity toward iodothyronine sulfates;
the soluble lysosomal ARSA and ARSB were inactive. Recombinant ARSC as
well as human placenta microsomes hydrolyzed iodothyronine sulfates with a
substrate preference for 3,3'-diiodothyronine sulfate (3,3'-T(2)S)
approximately T(3) sulfate (T(3)S) >> rT(3)S approximately T(4)S, whereas
human and rat liver microsomes showed a preference for 3,3'-T(2)S > T(3)S
>> rT(3)S approximately T(4)S. ARSC and the tissue microsomal sulfatases
were all characterized by high apparent K(m) values (>50 microM) for
3,3'-T(2)S and T(3)S. Iodothyronine sulfatase activity determined using
3,3'-T(2)S as a substrate was much higher in human liver microsomes than
in human placenta microsomes, although ARSC is expressed at higher levels
in human placenta than in human liver. The ratio of estrone sulfate to
T(2)S hydrolysis in human liver microsomes (0.2) differed largely from
that in ARSC homogenate (80) and human placenta microsomes (150). These
results suggest that ARSC accounts for the relatively low iodothyronine
sulfatase activity of human placenta, and that additional arylsulfatase(s)
contributes to the high iodothyronine sulfatase activity in human liver.
Further research is needed to identify these iodothyronine sulfatases, and
to study the physiological importance of the reversible sulfation of
iodothyronines in thyroid hormone metabolism
The metabolism and de-bromination of bromotyrosine in vivo
During inflammation, leukocyte-derived eosinophil peroxidase catalyses the formation of hypobromous acid, which can brominate tyrosine residues in proteins to form bromotyrosine. Since eosinophils are involved in the pathogenesis of allergic reactions, such as asthma, urinary bromotyrosine level has been used for the assessment of children with asthma. However, little is known about the metabolism and disposition of bromotyrosine in vivo. The aim of this study was to identify the major urinary metabolites formed during bromotyrosine metabolism and to develop mass spectrometric methods for their quantitation. Deuterium-labeled bromotyrosine was synthesized by deuterium exchange. [D3]bromotyrosine (500 nmole) was injected intraperitoneally into Sprague-Dawley rats and urine was collected for 24 h in a metabolic cage. 13C-labeled derivatives of bromotyrosine and its major urinary metabolite were synthesized and used as internal standards for quantitation. Following solid phase extraction, urine samples were derivatized to the pentafluorobenzyl ester, and analyzed using isotope dilution gas chromatography and negative-ion chemical ionization mass spectrometry. A novel brominated metabolite, 3-bromo-4-hydroxyphenylacetic acid (bromo-HPA), was identified as the major brominated metabolite of bromotyrosine. Bromo-HPA only accounted for 0.43Β±0.04% of infused [D3]bromotyrosine and 0.12Β±0.02% of infused [D3]bromotyrosine was excreted in the urine unchanged. However, ~1.3% (6.66Β±1.33 nmole) of infused [D3]bromotyrosine was excreted in the urine as the de-brominated metabolite, [D3]4-hydroxyphenylacetic acid, which is also a urinary metabolite of tyrosine in mammals. We also tested whether or not iodotyrosine dehalogenase can catalyse de-bromination of bromotyrosine and showed that iodotyrosine dehalogenase is able to de-brominate free bromotyrosine in vitro. We identified bromo-HPA as the main brominated urinary metabolite of bromotyrosine in rats. However, de-halogenation of bromotyrosine is the major metabolic pathway to eliminate free brominated tyrosine in vivo
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