Structural insights revealed by two novel THRB mutations.

PURPOSE: Among the inheritable forms of impaired sensitivity to thyroid hormone, resistance to thyroid hormone (RTH) due to mutations in the thyroid hormone receptor beta gene (THRB) is the first and best known described defect, revealing a wide phenotypic variability with an incompletely understood physiopathology. The objective of this study was to evaluate two novel mutations in THRB, N331H and L346R, in an attempt to provide a rational understanding of the harmful effects caused by them. METHODS: The mutations of two patients with RTHß were reproduced for analysis of gene transactivation by dual-luciferase reporter assay, and for molecular modeling for crystallography-based structural assessment. Serum measurements of TSH and FT4 were performed to compare the thyrotrophic resistance to thyroid hormone between RTHß patients and controls. RESULTS: Both mutants showed impaired gene transactivation, with greater damage in L346R. Molecular modeling suggested that the damage occurring in N331H is primarily due to reduced strength of the hydrogen bonds that stabilize T3 in its ligand-binding cavity (LBC), whereas in L346R, the damage is more marked and is mainly due to changes in hydrophobicity and molecular volume inside the LBC. Hormonal dosages indicated that the L346R mutant exhibited greater thyrotrophic resistance than N331H. CONCLUSIONS: This study provides a rational understanding of the effects of mutations, indicating deleterious structural changes in the LBC in both THR, and discloses that not only the position of the mutation but, notably, the nature of the amino acid exchange, has a cardinal role in the functional damage of the receptor.

Thermodynamic analysis of interactions of the Hsp90 with adenosine nucleotides: A comparative perspective.

Hsp90s are key proteins in cellular homeostasis since they interact with many client proteins. Several studies indicated that Hsp90s are potential targets for treating diseases, such as cancer or malaria. It has been shown that Hsp90s from different organisms have peculiarities despite their high sequence identity. Therefore, a detailed comparative analysis of several Hsp90 proteins is relevant to the overall understanding of their activity. Accordingly, the goal of this work was to evaluate the interaction of either ADP or ATP with recombinant Hsp90s from different organisms (human α and ß isoforms, Plasmodium falciparum, Leishmania braziliensis, yeast and sugarcane) by isothermal titration calorimetry. The measured thermodynamic signatures of those interactions indicated that despite the high identity among all Hsp90s, they have specific thermodynamic characteristics. Specifically, the interactions with ADP are driven by enthalpy but are opposed by entropy, whereas the interaction with ATP is driven by both enthalpy and entropy. Complimentary structural and molecular dynamics studies suggested that specific interactions with ADP that differ from those with ATP may contribute to the observed enthalpies and entropies. Altogether, the data suggest that selective inhibition may be more easily achieved using analogues of the Hsp90-ADP bound state than those of Hsp90-ATP bound state.

Crystallization and preliminary X-ray diffraction analysis of human IL-22 bound to its soluble decoy receptor IL-22BP.

Interleukin-22 (IL-22) is a pleiotropic cytokine that is involved in inflammatory responses. Human IL-22 was incubated with its soluble decoy receptor IL-22BP (IL-22 binding protein) and the IL-22-IL-22BP complex was crystallized in hanging drops using the vapour-diffusion method. Suitable crystals were obtained from polyethylene glycol solutions and diffraction data were collected to 2.75 A resolution. The crystal belonged to the tetragonal space group P4(1), with unit-cell parameters a = b = 67.9, c = 172.5 A, and contained two IL-22-IL-22BP complexes per asymmetric unit.