Harmonic oscillator model of the insulin and IGF1 receptors' allosteric binding and activation

The insulin and insulin-like growth factor 1 receptors activate overlapping signalling pathways that are critical for growth, metabolism, survival and longevity. Their mechanism of ligand binding and activation displays complex allosteric properties, which no mathematical model has been able to account for. Modelling these receptors' binding and activation in terms of interactions between the molecular components is problematical due to many unknown biochemical and structural details. Moreover, substantial combinatorial complexity originating from multivalent ligand binding further complicates the problem. On the basis of the available structural and biochemical information, we develop a physically plausible model of the receptor binding and activation, which is based on the concept of a harmonic oscillator. Modelling a network of interactions among all possible receptor intermediaries arising in the context of the model (35, for the insulin receptor) accurately reproduces for the first time all the kinetic properties of the receptor, and provides unique and robust estimates of the kinetic parameters. The harmonic oscillator model may be adaptable for many other dimeric/dimerizing receptor tyrosine kinases, cytokine receptors and G-protein-coupled receptors where ligand crosslinking occurs

Converting Insulin-like Growth Factors 1 and 2 into High-Affinity Ligands for Insulin Receptor Isoform A by the Introduction of an Evolutionarily Divergent Mutation

Insulin-like growth factors 1 and 2 (IGF-1 and -2, respectively) are protein hormones involved not only in normal growth and development but also in life span regulation and cancer. They exert their functions mainly through the IGF-1R or by binding to isoform A of the insulin receptor (IR-A). The development of IGF-1 and IGF-2 antagonists is of great clinical interest. Mutations of A4 and A8 sites of human insulin lead to disproportionate effects on hormone IR binding and activation. Here, we systematically modified IGF-1 sites 45, 46, and 49 and IGF-2 sites 45 and 48, which correspond, or are close, to insulin sites A4 and A8. The IGF-1R and IR-A binding and autophosphorylation potencies of these analogues were characterized. They retained the main IGF-1R-related properties, but the hormones with His49 in IGF-1 and His48 in IGF-2 showed significantly higher affinities for IR-A and for IR-B, being the strongest IGF-1- and IGF-2-like binders of these receptors ever reported. All analogues activated IR-A and IGF-1R without major discrepancies in their binding affinities. This study revealed that IR-A and IGF-1R contain specific sites, likely parts of their so-called sites 2′, which can interact differently with specifically modified IGF analogues. Moreover, a clear importance of IGF-2 site 44 for effective hormone folding was also observed. These findings may facilitate novel and rational engineering of new hormone analogues for IR-A and IGF-1R studies and for potential medical applications

Genome-wide gene expression profiling of testicular carcinoma in situ progression into overt tumours

The carcinoma in situ (CIS) cell is the common precursor of nearly all testicular germ cell tumours (TGCT). In a previous study, we examined the gene expression profile of CIS cells and found many features common to embryonic stem cells indicating that initiation of neoplastic transformation into CIS occurs early during foetal life. Progression into an overt tumour, however, typically first happens after puberty, where CIS cells transform into either a seminoma (SEM) or a nonseminoma (N-SEM). Here, we have compared the genome-wide gene expression of CIS cells to that of testicular SEM and a sample containing a mixture of N-SEM components, and analyse the data together with the previously published data on CIS. Genes showing expression in the SEM or N-SEM were selected, in order to identify gene expression markers associated with the progression of CIS cells. The identified markers were verified by reverse transcriptase–polymerase chain reaction and in situ hybridisation in a range of different TGCT samples. Verification showed some interpatient variation, but combined analysis of a range of the identified markers may discriminate TGCT samples as SEMs or N-SEMs. Of particular interest, we found that both DNMT3B (DNA (cytosine-5-)-methyltransferase 3 beta) and DNMT3L (DNA (cytosine-5-)-methyltransferase 3 like) were overexpressed in the N-SEMs, indicating the epigenetic differences between N-SEMs and classical SEM

Expression of the normal epithelial cell-specific 1 (NES1; KLK10) candidate tumour suppressor gene in normal and malignant testicular tissue

The normal epithelial cell-specific 1 (NES1) gene (official name kallikrein gene 10; KLK10) is a new member of the expanding human kallikrein gene family and encodes for a secreted serine protease. Experimental evidence suggests that NES1 controls normal cell growth and may function as a tumour suppressor. NES1 is down-regulated during breast cancer progression. The NES1 gene is highly expressed in testicular as well as in other tissues. In this study, we investigated the expression level of the NES1 gene in cancerous and normal testicular tissues with reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry. In all 14 primary testicular germ-cell tumours examined, the NES1 gene expression was markedly reduced compared to adjacent (paired) normal tissues. We further examined 6 randomly selected primary germ-cell tumours and 8 normal tissues (obtained from different individuals). We confirmed the differential expression of the NES1 gene in germ-cell tumours (GCT) and pre-malignant carcinoma in situ (CIS). Our findings suggest that NES1 may act as a tumour suppressor and may play a role in the pathogenesis and progression of this malignancy. © 2001 Cancer Research Campaign http://www.bjcancer.co

Aza-deoxycytidine induces apoptosis or differentiation via DNMT3B and targets embryonal carcinoma cells but not their differentiated derivatives

Background: Teratocarcinoma is a malignant male germ cell tumour, which contains stem cells and differentiated cancer tissues. DNMT3B has been shown to be highly expressed in human teratocarcinoma stem cells, and to mediate cytotoxicity of Aza-deoxycytidine (Aza-dC) in a pluripotent stem cell line NTERA2. Methods: We have established DNMT3B or POU5F1 (hereafter referred to as OCT4) knockdown in teratocarcinoma stem cells N2102Ep and TERA1 and in the pluripotent NTERA2 by a doxycycline-inducible system, and tested the cytotoxicity induced by Aza-dC. Results: Silencing of DNMT3B led to apoptosis of human teratocarcinoma stem cells N2102Ep and TERA1. Further, we found that induction of apoptosis or differentiation in NTERA2 and human embryonic stem cells by Aza-dC requires DNMT3B. To test whether Aza-dC inhibits proliferation of differentiated teratocarcinoma cells, we depleted OCT4 expression in N2102Ep and TERA1 cells treated with Aza-dC. Treatment with Aza-dC reduced cell number of differentiated cells to a lesser extent than their undifferentiated parental stem cells. Moreover, in contrast to the stem cells, Aza-dC failed to induce apoptosis of differentiated cells. Conclusions: Our finding suggests that DNMT3B acts as an antiapoptotic gene in teratocarcinoma stem cells, and mediates apoptosis and differentiation of human pluripotent stem cells induced by Aza-dC, and that Aza-dC specifically induces apoptosis of teratocarcinoma stem cells

Phenotypic variation within European carriers of the Y-chromosomal gr/gr deletion is independent of Y-chromosomal background.

BackgroundPrevious studies have compared sperm phenotypes between men with partial [1] deletions within the AZFc region of the Y chromosome with non-carriers, with variable results. Here, we have investigated a separate question, the basis of the variation in sperm phenotype within gr/gr deletion carriers, which ranges from normozoospermia to azoospermia. Differences in the genes removed by independent gr/gr deletions, the occurrence of subsequent duplications or the presence of linked modifying variants elsewhere on the chromosome have been suggested as possible causal factors. We set out to test these possibilities in a large sample of gr/gr deletion carriers with known phenotypes spanning the complete range.ResultsWe assembled a collection of 169 men diagnosed with gr/gr deletions from six centres in Europe and one in Australia, and characterized the DAZ and CDY1 copies retained, the presence or absence of duplications and the Y-chromosomal haplogroup. Although our study had good power to detect factors that accounted for 655.5% of the variation in sperm concentration, no such factor was detected. A negative effect of gr/gr deletions followed by b2/b4 duplication was observed within the normospermic group, which remains to be further explored in a larger study population. Finally, we observed significant geographical differences in the frequency of different subtypes of gr/gr deletions which may have relevance for the interpretation of case control studies dealing with admixed populations.ConclusionsWe conclude that the phenotypic variation of gr/gr carriers in men of European origin is largely independent of the Y-chromosomal background