HER3 (also known as c-ErbHuman epidermal growth factor receptor 3 (HER3) 1 (1) is a member of the type I receptor tyrosine kinase (RTK) family, which also includes EGFR, HER2/neu, and HER4 (2-4). HER3 forms heterodimers with other members of the type I RTK family, including the HER2/neu receptor (5-8). The HER2/neu receptor is amplified and overexpressed in 25-30% of human breast and 8 -11% of human ovarian cancers. This overexpression correlates with increased morbidity and mortality, and there is evidence that the overexpressed HER2 receptor leads to aggressive malignancies (9 -12). The HER2/HER3 heterodimer forms a high affinity heregulin receptor with tyrosine kinase activity. Heregulin binding to cells that display the HER2/HER3 heterodimer causes a mitogenic response both in vitro and in vivo, so understanding this interaction is of medical importance (13). The type I RTKs contain four extracellular domains, a single hydrophobic transmembrane segment, and a cytoplasmic tyrosine kinase domain (14). HER2/neu is a very active tyrosine kinase, but cells expressing HER2/neu alone, and not other members of the EGFR family, fail to bind heregulin. Conversely, the HER3 receptor binds heregulin but has low tyrosine kinase activity (15, 16). As mentioned above, the HER2/ HER3 heterodimer is a high affinity heregulin binding complex with signaling activity through the HER-2 kinase domain. To date, the domains of HER3 involved in ligand binding and heterodimerization have not been identified.Thus far, the high carbohydrate content (17) and the relatively large size (ϳ180 kDa) of the receptors in the EGFR family have hindered structural analysis by x-ray crystallography and NMR, so other methods have been sought to illuminate the structure and function of HER3. The extracellular domains (ECDs) of the type I RTKs have been divided into four domains: I, II, III, and IV, based on sequence analysis (18). Domains II and IV are cysteine-rich and are similar in sequence. Domains I and III also have sequence similarity (18,19). Little is known about the specific function of each domain except in EGFR, where several lines of evidence suggest that the major determinants for EGF binding lie in domain III. These lines of evidence include the following: 1) the exchange of domain III in chicken EGFR for domain III from human EGFR confers binding of human EGF (20, 21); 2) monoclonal antibodies that recognize residues in domain III prevent EGF binding to EGFR
In this report, we have used DNA Y-junctions as fluorescent scaffolds for EcoRII methyltransferase-thioredoxin (M.EcoRII-Trx) fusion proteins. Covalent links between the DNA scaffold and the methyltransferase were formed at preselected sites on the scaffold containing 5FdC. The resulting thioredoxin-targeted nanodevice was found to bind selectively to certain cell lines but not to others. The fusion protein was constructed so as to permit proteolytic cleavage of the thioredoxin peptide from the nanodevice. Proteolysis with thrombin or enterokinase effectively removed the thioredoxin peptide from the nanodevice and extinguished cell line specific binding measured by fluorescence. A number of potential applications for devices of this type can be envisioned. In particular, the ability of the fused protein to selectively target the nanodevice to certain tumor cell lines and not others suggests that this approach may serve as an adjunct to immunohistochemical methods in tumor classification as well as probe cell surface receptor architecture and function.
Bionanotechnology is an emerging field in nanotechnology. In general, it uses concepts from chemistry, biochemistry, and molecular biology to identify components and processes for the construction of self-assembling materials and devices. Distant goals of the science of bionanotechnology range from developing programmable nanoscale devices that can sample or alter their environments to developing assemblies capable of Darwinian evolution. At the heart of these approaches is the concept of the production of supramolecular assemblies (SMAs; also known as supramolecular aggregates) by programmed self-assembly in an aqueous medium. Ordered arrays, planar and closed-shell tilings, dynamic machines, and switches have been designed and constructed by using DNA-DNA, protein-protein, and protein-nucleic acid biospecificities. We review the designs and the analytical techniques that have been employed in the production of SMAs that do not occur in nature.
Purpose Potential acute exposure to ionizing radiation in nuclear or radiological accidents presents complex mass casualty scenarios that demand prompt triage and treatment decisions. Due to delayed symptoms and varied response of radiation victims, there is an urgent need to develop robust biomarkers to assess the extent of injuries in individuals. Experimental design The transcription factor Nrf2 is the master of redox homeostasis and there is transcriptional evidence of Nrf2‐dependent antioxidant response activation upon radiation. The biomarker potential of Nrf2‐dependent downstream target enzymes is investigated by measuring their response in bone marrow extracted from C57Bl/6 and C3H mice of both genders for up to 4 days following 6 Gy total body irradiation using targeted MS. Results Overall, C57Bl/6 mice have a stronger proteomic response than C3H mice. In both strains, male mice have more occurrences of upregulation in antioxidant enzymes than female mice. For C57Bl/6 male mice, three proteins show elevated abundances after radiation exposure: catalase, superoxide dismutase 1, and heme oxygenase 1. Across both strains and genders, glutathione S‐transferase Mu 1 is consistently decreased. Conclusions and clinical relevance This study provides the basis for future development of organ‐specific protein biomarkers used in diagnostic blood test for radiation injury.
Histological staining of reactive stroma has been shown to be a predictor of biochemical recurrence in prostate cancer, however, molecular markers of the stromal response to prostate cancer have not yet been fully delineated. The objective of this study was to determine whether or not the stromal biomarkers detected with a thioredoxin-targeted nanodevice could be used to distinguish the stroma associated with benign prostatic hyperplasia from that associated with PCA. In this regard, we recently demonstrated that a thioredoxin-targeted nanodevice selectively binds to reactive stroma in frozen prostate tumor tissue sections. To accomplish this, random frozen prostate tissue sections from each of 35 patients who underwent resection were incubated with the nanodevice and graded for fluorescent intensity. An adjacent section from each case was stained with Hematoxylin & Eosin to confirm the diagnosis. Select cases were stained with Masson's Trichrome or immunohistochemically using antibodies to thioredoxin reductase 1, thioredoxin reductase 2 or peroxiredoxin 1. Our results demonstrate that the graded intensity of nanodevice binding to the stroma associated with PCA was significantly higher (p = 0.0127) than that of benign prostatic hyperplasia using the t-test. Immunohistochemical staining of adjacent sections in representative cases showed that none of the two commonly studied thioredoxin interacting protein partners mirrored the fluorescence pattern seen with the nanodevice. However, thioredoxin reductase 2 protein was clearly shown to be a biomarker of prostate cancer-associated reactive stroma whose presence distinguishes the stroma associated with benign prostatic hyperplasia from that associated with prostate cancer. We conclude that the signal detected by the nanodevice, in contrast to individual targets detected with antibodies used in this study, originates from multiple thioredoxin interacting protein partners that distinguish the M2 neutrophil and macrophage associated inflammatory response in prostate cancer-associated stroma from the CD4+ T-Lymphocyte linked inflammation in benign prostatic hyperplasia.
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