Julie Rothacker

Ligand-induced dimer-tetramer transition during the activation of the cell surface epidermal growth factor receptor-A multidimensional microscopy analysis.

The epidermal growth factor receptor (EGFR) is a member of the erbB tyrosine kinase family of receptors. For many years it has been believed that receptor activation occurs via a monomer-dimer transition that is associated with a conformational change to activate the kinase. However, little is known about the quaternary state of the receptor at normal levels of expression (<105 receptors/cell). We employed multidimensional microscopy techniques to gain insight into the state of association of the human EGFR, in the absence and presence of ligand, on the surface of intact BaF/3 cells (50,000 receptors/cell). Image correlation microscopy of an EGFR-enhanced green fluorescent protein chimera was used to establish an average degree of aggregation on the submicron scale of 2.2 receptors/cluster in the absence of ligand increasing to 3.7 receptors/cluster in the presence of ligand. Energy transfer measurements between mixtures of fluorescein isothiocyanate-EGF and Alexa 555-EGF were performed using fluorescence lifetime imaging microscopy as a function of the donor: acceptor labeling ratio to gain insight into the spatial disposition of EGFR ligand binding sites on the nanometer scale. In the context of a two-state Förster resonance energy transfer (FRET)/non-FRET model, the data are consistent with a minimum transfer efficiency of 75% in the FRET population. The microscopy data are related to biophysical data on the EGFR in the A431 cell line and the three-dimensional structure of the ligated EGFR extracellular domain. In the context of a monomer-dimer-oligomer model, the biophysical data are consistent with a significant fraction of ligated EGFR tetramers comprising two dimers juxtaposed in a side-by-side (or slightly staggered) arrangement. Our data are consistent with a specific higher order association of the ligand-bound EGFR on the nanometer scale and indicate the existence of distinct signaling entities beyond the level of the EGFR dimer which could play an important role in receptor transactivation.

Proprotein convertases promote processing of VEGF-D, a critical step for binding the angiogenic receptor VEGFR-2

Vascular endothelial growth factor (VEGF)‐D is a secreted glycoprotein that induces angio‐genesis and lymphangiogenesis. It consists of a central domain, containing binding sites for VEGF receptor‐2 (VEGFR‐2) and VEGFR‐3, and N‐ and C‐terminal propep‐tides. It is secreted from the cell as homodimers of the full‐length form that can be proteolytically processed to remove the propeptides. It was recently shown, using adenoviral gene delivery, that fully processed VEGF‐D induces angiogenesis in vivo, whereas full‐length VEGF‐D does not. To better understand these observations, we monitored the effect of VEGF‐D processing on receptor binding using a full‐length VEGF‐D mutant that cannot be processed. This mutant binds VEGFR‐2, the receptor signaling for angiogenesis, with ~17, 000‐fold lower affinity than mature VEGF‐D, indicating the importance of processing for interaction with this receptor. Further, we show that members of the proprotein convertase (PC) family of proteases promote VEGF‐D processing, which facilitates the VEGF‐D/VEGFR‐2 interaction. The PCs furin and PC5 promote cleavage of both propeptides, whereas PC7 promotes cleavage of the C‐terminal propeptide only. The finding that PCs promote activation of VEGF‐D and other proteins with roles in cancer such as matrix metalloproteinases, emphasizes the importance of these enzymes as potential regulators of tumor progression and metastasis.—McColl, B. K., Paavonen, K., Karnezis, T., Harris, N. C., Davydova, N., Rothacker, J., Nice, E. C., Harder, K. W., Roufail, S., Hibbs, M. L., Rogers, P. A. W., Alitalo, K., Stacker, S. A., Achen, M. G. Proprotein convertases promote processing of VEGF‐D, a critical step for binding the angiogenic receptor VEGFR‐2. FASEB J. 21, 1088–1098 (2007)

Experimental determination of quantum dot size distributions, ligand packing densities, and bioconjugation using analytical ultracentrifugation.

Analytical ultracentrifugation (AUC) was used to characterize the size distribution and surface chemistry of quantum dots (QDs). AUC was found to be highly sensitive to nanocrystal size, resolving nanocrystal sizes that differ by a single lattice plane. Sedimentation velocity data were used to calculate the ligand packing density at the crystal surface for different sized nanocrystals. Dihydrolipoic acid poly(ethylene glycol) was found to bind between 66 and 60% of the surface cadmium atoms for CdSe nanocrystals in the 1.54-2.59 nm radius size regime. The surface ligand chemistry was found to affect QD sedimentation, with larger ligands decreasing the sedimentation rate through an increase in particle volume and increase in frictional coefficient. Finally, AUC was used to detect and analyze protein association to QDs. Addition of bovine serum albumin (BSA) to the QD sample resulted in a reduced sedimentation rate, which may be attributed to an associated frictional drag. We calculated that one to two BSA molecules bind per QD with an associated frictional ratio of 1.2.

Antibodies specifically targeting a locally misfolded region of tumor associated EGFR

Epidermal Growth Factor Receptor (EGFR) is involved in stimulating the growth of many human tumors, but the success of therapeutic agents has been limited in part by interference from the EGFR on normal tissues. Previously, we reported an antibody (mab806) against a truncated form of EGFR found commonly in gliomas. Remarkably, it also recognizes full-length EGFR on tumor cells but not on normal cells. However, the mechanism for this activity was unclear. Crystallographic structures for Fab:EGFR287–302 complexes of mAb806 (and a second, related antibody, mAb175) show that this peptide epitope adopts conformations similar to those found in the wtEGFR. However, in both conformations observed for wtEGFR, tethered and untethered, antibody binding would be prohibited by significant steric clashes with the CR1 domain. Thus, these antibodies must recognize a cryptic epitope in EGFR. Structurally, it appeared that breaking the disulfide bond preceding the epitope might allow the CR1 domain to open up sufficiently for antibody binding. The EGFRC271A/C283A mutant not only binds mAb806, but binds with 1:1 stoichiometry, which is significantly greater than wtEGFR binding. Although mAb806 and mAb175 decrease tumor growth in xenografts displaying mutant, overexpressed, or autocrine stimulated EGFR, neither antibody inhibits the in vitro growth of cells expressing wtEGFR. In contrast, mAb806 completely inhibits the ligand-associated stimulation of cells expressing EGFRC271A/C283A. Clearly, the binding of mAb806 and mAb175 to the wtEGFR requires the epitope to be exposed either during receptor activation, mutation, or overexpression. This mechanism suggests the possibility of generating antibodies to target other wild-type receptors on tumor cells.

Use of multiple reaction monitoring for multiplex analysis of colorectal cancer-associated proteins in human feces.

Colorectal cancer (CRC) is the second most common cause of cancer‐related deaths worldwide with an annual incidence of almost a million cases and an annual mortality around 500 000. The fecal occult blood test is currently the first line method for CRC screening, but has unacceptably low sensitivity and specificity. Improved screening tests are therefore urgently required for early‐stage CRC screening when therapy is most likely to be effective. We describe a discovery‐based proteomics hypothesis using orthogonal multi‐dimensional fractionation (1‐D SDS‐PAGE, RP‐HPLC, size exclusion chromatography) to mine deep into the fecal proteome for the initial discovery process, which generated a library containing 108 human fecal proteins with the associated peptide and MS/MS data. These data were then used to develop and optimize a multiplex multiple reaction monitoring assay for 40 non‐redundant human proteins present in the feces. To show proof of principal for clinical analysis, multiplex screening of these 40 proteins was carried out on fecal samples from eight CRC patient and seven normal volunteers. We identified 24 proteins consistently found in all samples and nine proteins found only in the CRC patients, showing the potential of this approach for the analysis of potential CRC biomarkers. Absolute quantitation using C‐terminal isotopically labeled synthetic peptides corresponding to hemoglobin and carcinoembryonic antigen 5 was also performed.

Murine fecal proteomics: a model system for the detection of potential biomarkers for colorectal cancer.

Tumor related products shed into the feces offer a potential source of biomarkers for the detection of colorectal cancer (CRC). Using SDS-PAGE followed by nanoflow reversed-phased LC-MS/MS to analyse fecal samples from Apc(Min/+) mice (that develop spontaneous multiple intestinal neoplasia with age) we have identified 336 proteins (115 proteins of murine origin, 201 from fecal bacteria, 18 associated with food intake and 2 of apparent parasitic origin). 75% of the murine proteins identified in this study are predicted to be extracellular or associated with the cell plasma membrane. Of these proteins, a number of the murine homologues of colorectal cancer associated proteins (CCAP) such as hemoglobin, haptoglobin, hemopexin, alpha-2-macroglobulin and cadherin-17 have been identified, demonstrating the potential of fecal proteomics for detecting potential biomarkers and paving the way for subsequent MS/MS based biomarker studies on similar human samples.

The propeptides of VEGF-D determine heparin binding, receptor heterodimerization and effects on tumor biology

Background: VEGF-D is a secreted protein that promotes cancer metastasis; it comprises a receptor-binding domain flanked by cleavable propeptides but the functions of the propeptides were unclear. Results: Propeptides determine heparin binding, VEGF receptor heterodimerization, and rates of metastasis. Conclusion: Propeptides influence molecular interactions of VEGF-D and its bioactivity in cancer. Significance: This study defines the biological significance of VEGF-D propeptides. VEGF-D is an angiogenic and lymphangiogenic glycoprotein that can be proteolytically processed generating various forms differing in subunit composition due to the presence or absence of N- and C-terminal propeptides. These propeptides flank the central VEGF homology domain, that contains the binding sites for VEGF receptors (VEGFRs), but their biological functions were unclear. Characterization of propeptide function will be important to clarify which forms of VEGF-D are biologically active and therefore clinically relevant. Here we use VEGF-D mutants deficient in either propeptide, and in the capacity to process the remaining propeptide, to monitor the functions of these domains. We report for the first time that VEGF-D binds heparin, and that the C-terminal propeptide significantly enhances this interaction (removal of this propeptide from full-length VEGF-D completely prevents heparin binding). We also show that removal of either the N- or C-terminal propeptide is required for VEGF-D to drive formation of VEGFR-2/VEGFR-3 heterodimers which have recently been shown to positively regulate angiogenic sprouting. The mature form of VEGF-D, lacking both propeptides, can also promote formation of these receptor heterodimers. In a mouse tumor model, removal of only the C-terminal propeptide from full-length VEGF-D was sufficient to enhance angiogenesis and tumor growth. In contrast, removal of both propeptides is required for high rates of lymph node metastasis. The findings reported here show that the propeptides profoundly influence molecular interactions of VEGF-D with VEGF receptors, co-receptors, and heparin, and its effects on tumor biology.

A truncated soluble epidermal growth factor receptor-Fc fusion ligand trap displays anti-tumour activity in vivo

A number of therapeutic strategies including small molecule tyrosine kinase inhibitors and monoclonal antibodies have been developed to target the epidermal growth factor receptor (EGFR) signalling axis for the treatment of cancer. To date, the focus of therapeutic intervention has been the EGFR itself. In the current study, we have assembled and expressed in mammalian cells a soluble, EGFR ligand trap comprising the first 501 amino acids of the mature EGFR sequence fused in-frame with a human IgG Fc domain. The fusion protein, designated sEGFR501.Fc, was secreted as a 220 kDa disulphide-linked homodimer that exhibited high affinity (0.4–8 nM) in competition assays for a number of EGFR ligands including EGF and transforming growth factor-α (TGF-α). sEGFR501.Fc inhibited EGF-stimulated tyrosine phosphorylation of the EGFR of the lung cancer cell lines A549 and H1437, and inhibited and blocked the proliferation of H1437 cells. Administration of sEGFR501.Fc to mice bearing human tumour xenografts derived from A431 (epidermoid carcinoma) and DU145 (androgen-independent prostate cancer) tumour cell lines resulted in modest retardation of tumour growth. These results provide proof-in-principle that using high affinity soluble receptors is a viable method for inhibiting multi-ligand systems, and the impetus to optimize this approach and develop reagents with greater affinity and broader specificity.

Ligand binding induces a conformational change in epidermal growth factor receptor dimers

The activation of the epidermal growth factor receptor (EGFR) kinase requires ligand binding to the extracellular domain (ECD). Previous reports demonstrate that the EGFR-ECD can be crystallized in two conformations – a tethered monomer or, in the presence of ligand, an untethered back-to-back dimer. We use Biosensor analysis to demonstrate that even in the monomeric state different C-terminal extensions of both truncated (EGFR1–501)-ECD and full-length EGFR1–621-ECD can change the conformation of the ligand-binding site. The binding of a monoclonal antibody mAb806, which recognizes the dimer interface, to the truncated EGFR1–501-Fc fusion protein is reduced in the presence of ligand, consistent with a change in conformation. On the cell surface, the presence of erythroblastosis B2 (erbB2) increases the binding of mAb806 to the EGFR. The conformation of the erbB2: EGFR heterodimer interface changes when the cells are treated with epidermal growth factor (EGF). We propose that ligand induces kinase-inactive, pre-formed EGFR dimers and heterodimers to change conformation leading to kinase-active tetramers, where kinase activation occurs via an asymmetric interaction between EGFR dimers.

Use of thiazolidine-mediated ligation for site specific biotinylation of mouse EGF for biosensor immobilisation

Biosensors provide a sophisticated and discriminating means of probing biomolecular interactions. Specific ligands such as peptides and proteins can be immobilized onto sensor surfaces by a number of means including covalent attachment via amine, thiol or aldehyde chemistry, capture via biotin-avidin interaction or the use of specific tags. We have devised a simple chemoselective ligation method to selectively conjugate an anchoring functionality onto N-terminal serine or threonine residues of peptides and proteins allowing them to be immobilised onto the sensor surface in a defined orientation. It is based on the specific reaction of the 1,2-aminothiol of cysteine with an aldehyde under acidic conditions to form a stable thiazolidine product. The carbonyl precursors are derived from the 1,2-aminoalcohols of Ser or Thr that can be selectively and rapidly converted to the aldehyde form by periodate oxidation. Biotinylation of the aldehyde is then achieved via simple conjugation with a novel water-soluble dipeptide that contains a lysine residue bearing an Nε-cysteine-derived 1,2-aminothiol and an Nα-biotin moiety. Use of this method allowed selective biotinylation of a native form of murine EGF (mEGF2-53) that has an N-terminal serine residue. This derivative was then immobilised onto a streptavidin biosensor surface, and the resultant surface activity compared with those obtained by immobilising recombinant human EGF or the soluble extracellular domain of the EGF receptor (sEGFR1-621) using amine coupling (NHS/EDC) chemistry. The surface recognised the recombinant sEGFR with a similar K D to that of human EGF immobilised using NHS/EDC chemistry, or if the receptor was immobilised and murine EGF injected.