Search

Ubiquitination controls the stability of most cellular proteins, and its deregulation contributes to human diseases including cancer. Deubiquitinases remove ubiquitin from proteins, and their inhibition can induce the degradation of selected proteins, potentially including otherwise undruggable targets. For example, the inhibition of ubiquitin-specific protease 7 (USP7) results in the degradation of the oncogenic E3 ligase MDM2, and leads to re-activation of the tumour suppressor p53 in various cancers. Here we report that two compounds, FT671 and FT827, inhibit USP7 with high affinity and specificity in vitro and within human cells. Co-crystal structures reveal that both compounds target a dynamic pocket near the catalytic centre of the auto-inhibited apo form of USP7, which differs from other USP deubiquitinases. Consistent with USP7 target engagement in cells, FT671 destabilizes USP7 substrates including MDM2, increases levels of p53, and results in the transcription of p53 target genes, induction of the tumour suppressor p21, and inhibition of tumour growth in mice., Author(s): Andrew P. Turnbull (corresponding author) [1]; Stephanos Ioannidis (corresponding author) [2]; Wojciech W. Krajewski [1]; Adan Pinto-Fernandez [3]; Claire Heride [4]; Agnes C. L. Martin [5]; Louise M. Tonkin [...]

Genetic studies have identified mutations in key regulators of the Wnt/[beta]-catenin pathway in a variety of cancers, most frequently in colon cancers. However, whether the pathway is activated in clinical cancer samples is not easily determined, and therefore it is useful to find markers that could be surrogates to show activation of the Wnt/[beta]-catenin pathway. Gene expression profiles were analyzed in SW620, a colon cancer cell line in which [beta]-catenin levels are stabilized as a consequence of truncated adenomatous polyposis coli and were compared with profiles of the same cells transfected with antisense oligodeoxynucleotides. Treatment of cells with [beta]-catenin antisense oligodeoxynucleotides resulted in a decrease in the levels of axin2 and human naked cuticle (hnkd) mRNAs. Interestingly, the proteins encoded by both of these mRNAs are known inhibitors of the [beta]-catenin pathway. In 30 human cell lines derived from different origins, axin2 and hnkd were expressed only in human colon cancer cell lines that are known to have activating mutations in the Wnt/[beta]-catenin pathway. Further, levels of both axin2 and hnkd mRNA were also found to be elevated in about 65% of laser microdissected cells from human colon tumors compared with laser microdissected cells of normal morphology from the same patient samples. The increased expression of axin2 and hnkd correlated with truncations in adenomatous polyposis coli in the same patient samples. These results reveal that it is possible to detect activation of a carcinogenic pathway in human cancer samples with specific markers.

The ob/ob mouse is genetically deficient in leptin and exhibits a phenotype that includes obesity and non-insulin-dependent diabetes melitus. This phenotype closely resembles the morbid obesity seen in humans. In this study, we demonstrate that a single intramuscular injection of a recombinant adeno-associated virus (AAV) vector encoding mouse leptin (rAAV-leptin) in ob/ob mice leads to prevention of obesity and diabetes. The treated animals show normalization of metabolic abnormalities including hyperglycemia, insulin resistance, impaired glucose tolerance, and lethargy. The effects of a single injection have lasted through the 6-month course of the study. At all time points measured the circulating levels of leptin in the serum were similar to age-matched control C57 mice. These results demonstrate that maintenance of normal levels of leptin (2-5 ng/ml) in the circulation can prevent both the onset of obesity and associated non-insulin-dependent diabetes. Thus a single injection of a rAAV vector expressing a therapeutic gene can lead to complete and long-term correction of a genetic disorder. Our study demonstrates the long-term correction of a disease caused by a genetic defect and proves the feasibility of using rAAV-based vectors for the treatment of chronic disorders like obesity.

Translocation of glucose transporter type 4 (GLUT4) occurs in mammalian cells with the help of platelet-derived growth factor (PDGF)-stimulated activity of phosphatidylinositol (PI) 3-kinase. A study of mutant PDGF receptors confirm the role of PI3-kinase in GLUT4 translocation. In 3T3-L1 adipocytes and chinese hamster ovary cells that overexpress PDGF receptors, GLUT4 translocation is induced by PDGF and insulin.

Fibroblast growth factor (FGF) receptor with a point mutation will no longer link with phospholipase C-gamma (PLC-gamma). This point mutation involves phenylalanine taking the place of tyrosine at residue 766. In addition, the mutant receptor will no longer regulate the hydrolysis of phosphatidylinositol (PtdIns) of the formation of Ca2+ following FGF stimulation. Hence the point mutation of the FGF receptor can sometimes stop activation of PLC-gamma. Moreover, FGF-induced mitogenesis can happen without Ca2+ mobilization or PtdIns hydrolysis.

WHEN PDGF BINDS TO ITS SPECIFIC receptor on the cell surface, the receptor protein becomes phosphorylated on tyrosine residues (autophosphorylated), and the conformation of the cytoplasmic domain of the receptor [...]

The tyrosine platelet-derived growth factor receptor-beta (PDGFR-beta) balances migration-promoting and migration-suppressing activities to maintain chemotaxis toward the PDGF-BB homodimer. Specific tyrosines are mapped to reveal the mechanism involved in regulating chemotaxis. Phosphatidylinositol-3-OH kinase promotes chemotaxis in association with PDGFR-beta while Ras-GTPase-activating protein inhibits migration.

The fms-like tyrosine kinase (Flt) is a transmembrane receptor in the tyrosine kinase family. Expression of flt complementary DNA in COS cells conferred specific, high-affinity binding of vascular endothelial growth factor, also known as vascular permeability factor (VEGF-VPF), a factor that induces vascular permeability when injected in the guinea pig skin and stimulates endothelial cell proliferation. Expression of Flt in Xenopus laevis oocytes caused the oocytes to release calcium in response to VEGF-VPF. These findings show that flt encodes a receptor for VEGF-VPF., Vascular permeability factor (VPF) was originally purified from guinea pig ascites and tumor cell culture medium as an agent that increases blood vessel permeability [1, 2]. VPF also stimulates migration [...]

Role of Phosphatidylinositol Kinase in PDGF Receptor Signal Transduction THE BINDING OF PDGF TO ITS RECEPTOR rapidly activates the receptor's tyrosine kinase and elicits a number of biochemical responses that [...]

A Common PDGF Receptor Is Activated by Homodimeric A and B Forms of PDGF PLATELET-DERIVED GROWTH FACTOR, a major mitogen for cells of mesenchymal origin, is a 32-kD protein heterodimer [...]