Biomedical subjects

Panagiota Iordanidou

Publications and source records attributed to Panagiota Iordanidou.

4 recordsLinked to original sources

PubMed2006

Functional characterization of hepatocyte nuclear factor-4 alpha dimerization interface mutants.

Hepatocyte nuclear factor-4 (HNF-4alpha), a member of the nuclear receptor superfamily, binds DNA exclusively as a homodimer. Dimerization controls important aspects of receptor function, such as DNA binding, protein stability, ligand binding and interaction with coactivators. Crystallographic data of the HNF-4alpha ligand-binding domain (LBD) demonstrated that the homodimer interface is composed of residues in helices 7, 9 and 10 with intermolecular salt bridges, hydrogen bonds and hydrophobic interactions contributing to the stability of the interface. To investigate the importance of the proposed ionic interactions for HNF-4alpha dimerization, interactions critical for formation of the LBD homodimer interface were disrupted by introducing point mutations in residues D261N (H7), E269Q (H7), Q307L (H9), D312N (H9) and Q336L (H10). Mutants were analysed for transactivation, coactivator interaction, DNA binding and dimerization. EMSA analysis showed that the mutants are able to bind DNA as dimers and coimmunoprecipitation assays confirmed dimerization in solution. Furthermore, the mutations do not compromise HNF-4alpha activity and are responsive to PPAR-gamma coactivator-1 (PGC-1). Finally, residue R324, located in the H9/H10 loop, which was suspected to be involved in dimer stabilization via an ionic interaction with residue E276, was studied. In contrast to the conservative substitution R324H the mutation R324L abolishes HNF-4alpha transcriptional activity and coactivator recruitment, revealing that the nature of substitution may play an important role in HNF-4alpha function.

Amino Acid Sequence↗

PubMed2005

Distinct amino acid residues may be involved in coactivator and ligand interactions in hepatocyte nuclear factor-4alpha.

Hepatocyte nuclear factor-4 (HNF-4) is a transcription factor of the nuclear hormone receptor superfamily that is constitutively active without the addition of exogenous ligand. Crystallographic analysis of the HNF-4alpha and HNF-4gamma ligand binding domains (LBDs) demonstrated the presence of endogenous ligands that may act as structural cofactors for HNF-4. It was also proposed by crystallographic studies that a combination of ligand and coactivator might be required to lock the receptor in its active state. We previously showed that mutations in amino acid residues Ser-181 and Met-182 in H3, Leu-219 and Leu-220 and Arg-226 in H5, Ileu-338 in H10, and Ileu-346 in H11, which line the LBD pocket in HNF-4alpha and come in contact with the ligand, impair its transactivation potential. In the present study, physical and functional interaction assays were utilized with two different coactivators, PGC-1 and SRC-3, to address the role of coactivators in HNF-4 function. We show that the integrity of the hinge (D) domain of HNF-4alpha and the activation function (AF)-2 activation domain region are critical for coactivation. Surprisingly, a different mode of coactivation is observed among the LBD point mutants that lack transcriptional activity. In particular, coactivation is maintained in mutants Ser-181, Arg-226, and Ile-346 but is abolished in mutants Met-182, Leu-219, and Ile-338. Physical interactions confirm this pattern of activation, implying that distinct amino acid residues may be involved in coactivator and ligand interactions, although some residues may be critical for both functions. Our results provide evidence and expand predictions based on the crystallographic data as to the role of coactivators in HNF-4alpha constitutive transcriptional activity.

Amino Acids↗

PubMed2004

Critical role of residues defining the ligand binding pocket in hepatocyte nuclear factor-4alpha.

Hepatocyte nuclear factor-4alpha (HNF-4alpha), a member of the nuclear receptor superfamily, is a crucial regulator of a large number of genes involved in glucose, cholesterol, and fatty acid metabolism. Unlike other members of the superfamily, HNF-4alpha activates transcription in the absence of exogenously added ligand. Recently published crystallographic data show that fatty acids are endogenous ligands for HNF-4. Transcriptional analysis of point mutations of the residues that are located in helices H3, H5, H10, and H11, which have been shown to come in contact with the ligand, resulted in a dramatic decrease in activity, without affecting DNA binding and dimerization. Our results show the importance of residues Ser-181, Met-182 in H3, Leu-219, Leu-220 and Arg-226 in H5, Ile-338 in H10, and Ile-346 in H11 that line the ligand-binding domain pocket in HNF-4alpha and impair its transactivation potential. Structural modeling reveals that the mutations do not cause any large scale structural alterations, and the observed loss in transactivation can be attributed to local changes, demonstrating that these residues play a significant role in maintaining the structural integrity of the HNF-4alpha ligand binding pocket.

Amino Acid Sequence↗

PubMed2002

A novel immunoglobulin superfamily receptor (19A) related to CD2 is expressed on activated lymphocytes and promotes homotypic B-cell adhesion.

A novel lymphocyte-specific immunoglobulin superfamily protein (19A) has been cloned. The predicted 335-amino-acid sequence of 19A represents a Type 1 membrane protein with homology with the CD2 family of receptors. A molecular model of the two predicted extracellular immunoglobulin-like domains of 19A has been generated using the crystal structure of CD2 as a template. In isolated lymphocytes, expression of 19A is induced by various activation stimuli, and enforced expression of the 19A gene promotes homotypic cell adhesion in a B-cell-line model. Collectively these data imply that the 19A protein plays a role in regulation of lymphocyte adhesion.

Amino Acid Sequence↗