Modeling the Epidermal Growth Factor—Epidermal Growth Factor Receptor L2 Domain Interaction: Implications for the Ligand Binding Process

Abstract

Signaling from the epidermal growth factor (EGF) receptor is triggered by the binding of lig-ands such as EGF or transforming growth factor alpha (TGF-α) and subsequent receptor dimerization. An understanding of these processes has been hindered by the lack of structural information about the ligand-bound, dimerized EGF receptor. Using an NMR-derived structure of EGF and a homology model of the major ligand binding domain of the EGF receptor and experimental data, we modeled the binding of EGF to this EGF receptor fragment. In this low resolution model of the complex, EGF sits across the second face of the EGF receptor L2 domain and EGF residues 10–16, 36–37, 40–47 bind to this face. The structural model is largely consistent with previously published NMR data describing the residues of TGF-α which interact strongly with the EGF receptor. Other EGF residues implicated in receptor binding are accounted by our proposal that the ligand binding is a two-step process with the EGF binding to at least one other site of the receptor. This three-dimensional model is expected to be useful in the design of ligand-based antagonists of the receptor.     

Discovery of novel 1,4-dihydropyridine-based PDE4 inhibitors

Substituted 1,4-dihydropyridines were discovered as a novel and potent series of phosphodiesterase 4 (PDE4) inhibitors. Structure–activity relationships within this series have been carried out and studies revealed that the dihydropyridine core, with indole moiety and 3,4-dimethoxybenzyl group, is a potent analogue for PDE4 inhibition. These novel series of compounds were prepared via a 3-component reaction in a single pot. In vitro biological activity, modeling studies and crystallography data are also reported.     

Arylglycine derivatives as potent transient receptor potential melastatin 8 (TRPM8) antagonists

A series of arylglycine-based analogs was synthesized and tested for TRPM8 antagonism in a cell-based functional assay. Following structure–activity relationship studies in vitro, a number of compounds were identified as potent TRPM8 antagonists and were subsequently evaluated in an in vivo pharmacodynamic assay of icilin-induced ‘wet-dog’ shaking in which compound 12 was fully effective. TRPM8 antagonists of the type described here may be useful in treating pain conditions wherein cold hypersensitivity is a dominant feature.     

Capture and display of antibodies secreted by hybridoma cells enables fluorescent on-cell screening

Hybridoma methods for monoclonal antibody (mAb) cloning are a mainstay of biomedical research, but they are hindered by the need to maintain hybridomas in oligoclonal pools during antibody screening. Here, we describe a system in which hybridomas specifically capture and display the mAbs they secrete: On-Cell mAb Screening (OCMS?). In OCMS?, mAbs displayed on the cell surface can be rapidly assayed for expression level and binding specificity using fluorescent antigens with high-content (image-based) methods or flow cytometry. OCMS? demonstrated specific mAb binding to poliovirus and rabies virus by forming a cell surface IgG “cap”, as a universal assay for anti-viral mAbs. We produced and characterized OCMS?-enabled hybridomas secreting mAbs that neutralize poliovirus and used fluorescence microscopy to identify and clone a human mAb specific for the human N-methyl-D-aspartate receptor. Lastly, we used OCMS? to assess expression and antigen binding of a recombinant mAb produced in 293T cells. As a novel method to physically associate mAbs with the hybridomas that secrete them, OCMS? overcomes a central challenge to hybridoma mAb screening and offers new paradigms for mAb discovery and production.     

Impact of P-Site tRNA and Antibiotics on Ribosome Mediated Protein Folding: Studies Using the Escherichia coli Ribosome

Background

The ribosome, which acts as a platform for mRNA encoded polypeptide synthesis, is also capable of assisting in folding of polypeptide chains. The peptidyl transferase center (PTC) that catalyzes peptide bond formation resides in the domain V of the 23S rRNA of the bacterial ribosome. Proper positioning of the 3′ –CCA ends of the A- and P-site tRNAs via specific interactions with the nucleotides of the PTC are crucial for peptidyl transferase activity. This RNA domain is also the center for ribosomal chaperoning activity. The unfolded polypeptide chains interact with the specific nucleotides of the PTC and are released in a folding competent form. In vitro transcribed RNA corresponding to this domain (bDV RNA) also displays chaperoning activity.

Results

The present study explores the effects of tRNAs, antibiotics that are A- and P-site PTC substrate analogs (puromycin and blasticidin) and macrolide antibiotics (erythromycin and josamycin) on the chaperoning ability of the E. coli ribosome and bDV RNA. Our studies using mRNA programmed ribosomes show that a tRNA positioned at the P-site effectively inhibits the ribosome''s chaperoning function. We also show that the antibiotic blasticidin (that mimics the interaction between 3′–CCA end of P/P-site tRNA with the PTC) is more effective in inhibiting ribosome and bDV RNA chaperoning ability than either puromycin or the macrolide antibiotics. Mutational studies of the bDV RNA could identify the nucleotides U2585 and G2252 (both of which interact with P-site tRNA) to be important for its chaperoning ability.

Conclusion

Both protein synthesis and their proper folding are crucial for maintenance of a functional cellular proteome. The PTC of the ribosome is attributed with both these abilities. The silencing of the chaperoning ability of the ribosome in the presence of P-site bound tRNA might be a way to segregate these two important functions.     

Platform Comparison for Evaluation of ALK Protein Immunohistochemical Expression,Genomic Copy Number and Hotspot Mutation Status in Neuroblastomas

ALK is an established causative oncogenic driver in neuroblastoma, and is likely to emerge as a routine biomarker in neuroblastoma diagnostics. At present, the optimal strategy for clinical diagnostic evaluation of ALK protein, genomic and hotspot mutation status is not well-studied. We evaluated ALK immunohistochemical (IHC) protein expression using three different antibodies (ALK1, 5A4 and D5F3 clones), ALK genomic status using single-color chromogenic in situ hybridization (CISH), and ALK hotspot mutation status using conventional Sanger sequencing and a next-generation sequencing platform (Ion Torrent Personal Genome Machine (IT-PGM)), in archival formalin-fixed, paraffin-embedded neuroblastoma samples. We found a significant difference in IHC results using the three different antibodies, with the highest percentage of positive cases seen on D5F3 immunohistochemistry. Correlation with ALK genomic and hotspot mutational status revealed that the majority of D5F3 ALK-positive cases did not possess either ALK genomic amplification or hotspot mutations. Comparison of sequencing platforms showed a perfect correlation between conventional Sanger and IT-PGM sequencing. Our findings suggest that D5F3 immunohistochemistry, single-color CISH and IT-PGM sequencing are suitable assays for evaluation of ALK status in future neuroblastoma clinical trials.     

Enhancement of adipogenic and osteogenic differentiation of human bone-marrow-derived mesenchymal stem cells by supplementation with umbilical cord blood serum

Umbilical cord blood serum (UCBS) is a promising replacement for animal sera for the culture of human mesenchymal stem cells (hMSC), the unique serum composition of UCBS appearing to have variable effects on their proliferation and differentiation. Conditioning UCBS with methods such as charcoal stripping assists specific processes such as adipogenesis and osteogenesis in hMSCs. The charcoal stripping of serum removes lipophilic materials such as oestrogens, which are known inhibitors of adipogenesis. hMSC cultures supplemented with charcoal-stripped UCBS (CS-UCBS) show enhanced adipogenesis in adipogenic induction medium (AIM) containing indomethacin, 3-isobutyl-1-methylxanthine and dexamethasone. To obtain efficient adipogenesis without CS-UCBS, we have developed a modified protocol in which cells cultured separately with UCBS and CS-UCBS are constantly treated with minimal doses of insulin (1.1 μg/ml) for 10 days prior to the addition of AIM. hMSC cultures differentiated by using the modified protocol show improved adipogenesis under fetal bovine serum (FBS), UCBS and CS-UCBS conditions, with levels of adipogenesis being highest in UCBS, thereby eliminating the need for charcoal stripping. Furthermore, in each of the three sera, the insulin-pre-treated hMSCs accumulate lipid droplets faster and exhibit improved adipogenesis overall when compared with normal AIM-induced adipogenesis. We have also compared the levels of osteogenesis in hMSCs by using an induction medium devoid of dexamethasone. Maximum calcium deposition has been observed in hMSCs cultured with UCBS, as compared with those cultured with FBS or CS-UCBS. Our newly developed methods with a humanized serum supplement thus enhance the differentiation of cultured hMSCs.     

Localization of the membrane-anchored MMP-regulator RECK at the neuromuscular junctions

Nerve apposition on nicotinic acetylcholine receptor clusters and invagination of the post-synaptic membrane (i.e. secondary fold formation) occur by embryonic day 18.5 at the neuromuscular junctions (NMJs) in mouse skeletal muscles. Finding the molecules expressed at the NMJ at this stage of development may help elucidating how the strong linkage between a nerve terminal and a muscle fiber is established. Immunohistochemical analyses indicated that the membrane-anchored matrix metalloproteinase regulator RECK was enriched at the NMJ in adult skeletal muscles. Confocal and electron microscopy revealed the localization of RECK immunoreactivity in secondary folds and subsynaptic intracellular compartments in muscles. Time course studies indicated that RECK immunoreactivity becomes associated with the NMJ in the diaphragm at around embryonic day 18.5 and thereafter. These findings, together with known properties of RECK, support the hypothesis that RECK participates in NMJ formation and/or maintenance, possibly by protecting extracellular components, such as synaptic basal laminae, from proteolytic degradation.     

Isotopic evidence of partial mycoheterotrophy in Burmannia coelestis (Burmanniaceae)

The Burmanniaceae contain several lineages of achlorophyllous mycoheterotrophic plants that associate with arbuscular mycorrhizal fungi (AMF). Here we investigate the isotopic profile of a green and potentially mycoheterotrophic plant in situ, Burmannia coelestis, and associated reference plants. We generated δ ¹³C and δ ¹⁵N stable isotope profiles for five populations of B. coelestis. Burmannia coelestis was significantly enriched in ¹³C relative to surrounding C₃ reference plants and significantly depleted in ¹³C relative to C₄ reference plants. No significant differences were detected in ¹⁵N enrichment between B. coelestis and reference plants. The isotopic profiles measured are suggestive of partial mycoheterotrophy in B. coelestis. Within the genus Burmannia transitions to full mycoheterotrophy have occurred numerous times, suggesting that some green Burmannia species are likely to be partially mycoheterotrophic but in many conditions this mode of nutrition may only be detectable using natural abundance stable isotopic methods, such as when associated with C₄ mycorrhizal plants.