Effect of Selenium on HLA-DR Expression of Thyrocytes.

Autoimmune thyroid diseases (ATDs) represent the most frequent forms of the organ-specific autoimmune thyroid disorders that result from interaction between genetic and environmental factors. Selenium has been shown to exert a beneficial effect on the autoimmune thyroiditis. In spite of therapeutical effect of selenium on autoimmunity, the mechanism of its action has not been revealed. Objective. To determine whether selenium in vitro thyrocytes cultures are able to influence the HLA-DR molecule expression of human thyrocytes and production of free oxygen radicals. Method. Thyrocytes were prepared from human thyroid gland and cultured in vitro in the presence of interferon-γ and sodium selenite. The expression of HLA-DR molecules induced by interferon-γ in the presence of sodium selenite of various concentration was measured by fluorescence-activated cell sorter. Results. Selenium has a dose-dependent inhibitory effect on the expression of HLA-DR molecules of thyrocytes induced by interferon-γ. This effect of selenium was in the inverse correlation with antioxidative capacity. Conclusion. Beneficial effect of selenium on autoimmune mechanism is a complex mechanism in which the inhibitory effect on HLA-DR molecule expression and antioxidative capacity are involved into therapy of autoimmune thyroiditis.

Cleavage of the human thyrotropin receptor by ADAM10 is regulated by thyrotropin.

The thyrotropin receptor (TSHR) has a unique 50 residue (317-366) ectodomain insertion that sets it apart from other glycoprotein hormone receptors (GPHRs). Other ancient members of the leucine-rich repeat G protein-coupled receptor (GPCR) (LGR) family do exhibit ectodomain insertions of variable lengths and sequences. The TSHR-specific insert is digested, apparently spontaneously, to release the ectodomain (A-subunit) leaving the balance of the ectodomain attached to the serpentine (B-subunit). Despite concerted efforts for the last 12 years by many laboratories, the enzyme involved in TSHR cleavage has not been identified and a physiologic role for this process remains unclear. Several lines of evidence had suggested that the TSHR protease is likely a member of the a disintegrin and metalloprotease (ADAM) family of metalloproteases. We show here that the expression of ADAM10 was specific to the thyroid by specially designed DNA microarrays. We also show that TSH increases TSHR cleavage in a dose-dependent manner. To prove that ADAM10 is indeed the TSHR cleavage enzyme, we investigated the effect of TSH-induced cleavage by a peptide based on a motif (TSHR residues 334-349), shared with known ADAM10 substrates. TSH increased dose dependently TSHR ectodomain cleavage in the presence of wild-type peptide but not a scrambled control peptide. Interestingly, TSH increased the abundance of non-cleaved single chain receptor, as well higher molecular forms of the A-subunit, despite their enhancement of the appearance of the fully digested A-subunit. This TSH-related increase in TSHR digested forms was further increased by wild-type peptide. We have identified for the first time ADAM10 as the TSHR cleavage enzyme and shown that TSH regulates its activation.

Non-random features of loop-size chromatin fragmentation.

Upon isolation of DNA from normal eukaryotic cells by standard methods involving extensive proteolytic treatment, a rather homogeneous population of loop-size, double-stranded DNA fragments is regularly obtained. These DNA molecules can be efficiently end-labeled by the DNA polymerase I Klenow fragment, as well as by a 3'- to -5'-exonuclease-free Klenow enzyme, but not by terminal transferase (TdT) unless the ends have been filled up by Klenow, suggesting that dominantly 5' protruding termini are generated upon fragmentation. The filled-up termini were used for cloning the distal parts of the approximately 50 kb fragments. BLAST analysis of the sequence of several clones allowed us to determine the sequence of the non-cloned side of the breakpoints. Comparison of 25, 600 bp-long breakpoint sequences demonstrated prevalence of repetitive elements. Consensus motives characteristic of the breakpoint sequences have been identified. Several sequences exhibit peculiar computed conformational characteristics, with sharp transition or center of symmetry located exactly at the breakpoint. Our data collectively suggest that chromatin fragmentation involves nucleolytic cleavages at fragile/hypersensitive sites delimiting loop-size fragments in a non-random manner. Interestingly, the sequence characteristics of the breakpoints are reminiscent of certain breakpoint cluster regions frequently subject to gene rearrangements.

Evolution of the thyrotropin receptor: a G protein coupled receptor with an intrinsic capacity to dimerize.

The rapidly escalating number of genome sequences has emphasized the basic tenants of the schema of life. By the same token comparisons according to specialized function or niche within nature expose genomic strategies to optimize the use of resources and ensure biological success. Increasing complexity may result from diversification, shuffling, and re-arrangement of an otherwise limited functional genomic complement. To further test the concept of relative structural plasticity of the TSH receptor we sequenced the TSHR gene of two Old World monkey species Macaca mulatta and Cercopithecus aethiops, evolutionary removed from Homo sapiens by >20Myr. Both genes encoded a protein of 764 residues. This structure was 99% homologous between the two species of Old World monkeys while C. aethiops was 97% and M. mulatta was 96% homologous to H. sapiens. TSHR sequence comparisons were sought for an additional eight mammals as well as four (two Salmon, Tilapia, and Sea Bass) from teleosts. The amino-acid sequences of the 14 TSH receptors were similar. The most variable sequences were those of the intracellular tail and the distal cysteine-rich C-terminus flanking region of the ectodomain, whereas the trans-membrane domain was most preserved. Some sequences were decidedly H. sapiens specific, while others were primate specific or showed the changes expected of evolutionary descent. Others, however, exhibited "cross-species polymorphism," sometimes at quite remarkable evolutionary distances. As opposed to H. sapiens the sequence differences may have subtle influences on TSHR function or may affect long-range compensation for radical changes in adducts. The two Old World monkeys share with other lower mammals the absence of a glycosylation site at 113-115. Sea Bass and Tilapia have four glycosylation sites, whereas the two salmon receptors have only three. Changes in some critical residues raise questions about variation in function: thus S281 is conserved in all mammals and an important determinant of negative agonist function of TSHR is replaced by R in Sea Bass. Likewise the K183, found at an important transitional region at LRR 6 conserved in all mammals, is represented by M in fish and may contribute to TSHR lutenization in fish. There is no evidence that evolutionary changes in primate receptors are more rapid than that in other mammals and the separation times of different mammals based on silent nucleotide changes of TSHR are closely parallel to archaeological estimates. Results of correlated mutation analysis, referenced to the rhodopsin crystal structure, affirms dimerization of TSHR transmembrane helices. In addition, it suggests the involvement of critical lipid-facing residues in the helices in receptor dimerization and oligomerization. We highlight the value of evolutionary informatics and set the stage for dissecting out potential subtle differences in TSHR function associated with structural variations.