Erik Helmerhorst

A comparative structural bioinformatics analysis of the insulin receptor family ectodomain based on phylogenetic information

The insulin receptor (IR), the insulin-like growth factor 1 receptor (IGF1R) and the insulin receptor-related receptor (IRR) are covalently-linked homodimers made up of several structural domains. The molecular mechanism of ligand binding to the ectodomain of these receptors and the resulting activation of their tyrosine kinase domain is still not well understood. We have carried out an amino acid residue conservation analysis in order to reconstruct the phylogeny of the IR Family. We have confirmed the location of ligand binding site 1 of the IGF1R and IR. Importantly, we have also predicted the likely location of the insulin binding site 2 on the surface of the fibronectin type III domains of the IR. An evolutionary conserved surface on the second leucine-rich domain that may interact with the ligand could not be detected. We suggest a possible mechanical trigger of the activation of the IR that involves a slight ‘twist’ rotation of the last two fibronectin type III domains in order to face the likely location of insulin. Finally, a strong selective pressure was found amongst the IRR orthologous sequences, suggesting that this orphan receptor has a yet unknown physiological role which may be conserved from amphibians to mammals.

jAMVLE, A new integrated molecular visualization learning environment*

A new computer‐based molecular visualization tool has been developed for teaching, and learning, molecular structure. This java‐based jmol Amalgamated Molecular Visualization Learning Environment (jAMVLE) is platform‐independent, integrated, and interactive. It has an overall graphical user interface that is intuitive and easy to use. The application can be downloaded free from the internet at wabri.org.au/jamvle. A cohort of 28 third year undergraduate molecular biotechnology degree students evaluated the new application through an essay‐style project. These were analyzed to identify themes expressed by students in the content of their evaluations. Most students were positive about the new jAMVLE learning environment, and five major benefits emerged from the analysis. In particular, the students perceived that jAMVLE has an appealing interface, is interactive, provides a useful integrated environment, is user friendly, and is an excellent learning tool. Overall, students found that the jAMVLE application stimulated their interest, was a more active learning environment, provided better guidance, and made learning fun.

The self‐association of HMGB1 and its possible role in the binding to DNA and cell‐membrane receptors

High mobility group box 1 (HMGB1), a chromatin protein, interacts with DNA and controls gene expression. However, when HMGB1 is released from apoptotic or damaged cells, it triggers proinflammatory reactions by interacting with various receptors, mainly receptor for advanced glycation end‐products (RAGE) and toll‐like receptors (TLRs). The self‐association of HMGB1 has been found to be crucial for its DNA‐related biological functions. It is influenced by several factors, such as ionic strength, pH, specific divalent metal cations, redox environment and acetylation. This self‐association may also play a role in the interaction with RAGE and TLRs and the concomitant inflammatory responses. Future studies should address the potential role of HMGB1 self‐association on its interactions with DNA, RAGE and TLRs, as well as the influence of physicochemical factors in different cellular environments on these interactions.

Insulin decreases the secretion of apoB-100 from hepatic HepG2 cells but does not decrease the secretion of apoB-48 from intestinal CaCo-2 cells.

We compared the acute effect of insulin on the human colonic intestinal epithelial cell line CaCo-2 and the transformed human hepatic cell line HepG2. Over 24 h, 100 nM and 10 microM insulin significantly inhibited the secretion of apolipoprotein (apo) B-100 from HepG2 cells to 63 and 49% of control, respectively. Insulin had no effect on the secretion of apoB-48 from CaCo-2 cells. There was no effect of insulin on the cholesterol ester or free cholesterol concentrations in HepG2 or CaCo-2 cells. HepG2 and CaCo-2 cells bound insulin with high affinity, leading to similar stimulation of insulin receptor protein tyrosine kinase activation. Protein kinase C or mitogen-activated protein kinase activity in the presence or absence of insulin was not correlated with apoB-48 production in CaCo-2 cells. Therefore, insulin acutely decreases the secretion of apoB-100 in hepatic HepG2 cells, but does not acutely modulate the production or secretion of apoB-48 from CaCo-2 intestinal cells.

The effect of physicochemical factors on the self-association of HMGB1: A surface plasmon resonance study

HMGB1 triggers proinflammatory reactions by interacting extracellularly with various receptors. HMGB1 also acts in the nucleus by interacting with DNA and controlling DNA transcription, a process which involves its self-association. The self-association of HMGB1 was characterized using surface plasmon resonance (SPR). A dimer/tetramer binding model was developed that provided a good fit to the SPR sensorgrams and enabled the kinetics of self-association of different HMGB1 oligomers to be evaluated under a variety of physicochemical conditions. The formation of HMGB1 tetramers, and not dimers, was strongly influenced by ionic strength. HMGB1 self-association increased as the pH was decreased from 7.4 to 4.8 but was abolished at pH4.0, suggesting the involvement of acidic amino acids of HMGB1 in its self-association. HMGB1 dimers were found to predominate in the absence of zinc, but addition of zinc promoted the formation of HMGB1 tetramers. More reducing conditions favored dimerization but diminished tetramer formation. In contrast, oxidizing conditions favored tetramer formation. Physicochemical factors modulate the extent of self-association of HMGB1. We speculate that HMGB1 dimers may preferentially bind DNA, whereas HMGB1 tetramers may promote inflammatory responses by binding to RAGE and TLRs. The self-association of HMGB1, regulated by variations of physicochemical factors, may influence its roles in DNA rearrangement and regulation of pathophysiological diseases.