Deubiquitination of EGFR by Cezanne-1 contributes to cancer progression.

Once stimulated, the epidermal growth factor receptor (EGFR) undergoes self-phosphorylation, which, on the one hand, instigates signaling cascades, and on the other hand, recruits CBL ubiquitin ligases, which mark EGFRs for degradation. Using RNA interference screens, we identified a deubiquitinating enzyme, Cezanne-1, that opposes receptor degradation and enhances EGFR signaling. These functions require the catalytic- and ubiquitin-binding domains of Cezanne-1, and they involve physical interactions and transphosphorylation of Cezanne-1 by EGFR. In line with the ability of Cezanne-1 to augment EGF-induced growth and migration signals, the enzyme is overexpressed in breast cancer. Congruently, the corresponding gene is amplified in approximately one third of mammary tumors, and high transcript levels predict an aggressive disease course. In conclusion, deubiquitination by Cezanne-1 curtails degradation of growth factor receptors, thereby promotes oncogenic growth signals.

EGFRvIV: a previously uncharacterized oncogenic mutant reveals a kinase autoinhibitory mechanism.

Tumor cells often subvert normal regulatory mechanisms of signal transduction. This study shows this principle by studying yet uncharacterized mutants of the epidermal growth factor receptor (EGFR) previously identified in glioblastoma multiforme, which is the most aggressive brain tumor in adults. Unlike the well-characterized EGFRvIII mutant form, which lacks a portion of the ligand-binding cleft within the extracellular domain, EGFRvIVa and EGFRvIVb lack internal segments distal to the intracellular tyrosine kinase domain. By constructing the mutants and by ectopic expression in naive cells, we show that both mutants confer an oncogenic potential in vitro, as well as tumorigenic growth in animals. The underlying mechanisms entail constitutive receptor dimerization and basal activation of the kinase domain, likely through a mechanism that relieves a restraining molecular fold, along with stabilization due to association with HSP90. Phosphoproteomic analyses delineated the signaling pathways preferentially engaged by EGFRvIVb-identified unique substrates. This information, along with remarkable sensitivities to tyrosine kinase blockers and to a chaperone inhibitor, proposes strategies for pharmacological interception in brain tumors harboring EGFRvIV mutations.

The use of circular stapler for cervical esophagogastric anastomosis after esophagectomy: surgical technique and early postoperative outcome.

Stapled esophagogastric anastomosis after esophagectomy is considered to be superior to traditional handsewn techniques. Linear staplers are usually used. The aim of this study is to evaluate early postoperative results of circular stapler in cervical esophagogastric anastomosis. Records of all patients who underwent esophagectomy during the years 2003-2008 were reviewed. Patients that underwent transthoracic esophagectomy, colon transposition, or linear stapler anastomosis were excluded. Esophagogastric anastomosis was done either handsewn or using circular stapler. Patients underwent either pyloromyotomy, pyloroplasty, or no pyloric intervention. Postoperative leakage was diagnosed either clinically or radiologically. The end-point of this study was the incidence of anastomotic leak in the immediate postoperative period. Eighty-two patients (average age 66 years, male/female, 52/30) met the inclusion criteria. In 30 patients, the anastomosis was handsewn, and in 52 patients, it was done using a circular stapler. Overall operative mortality rate was 4.8% (four patients because of pulmonary or cardiac complications). Anastomotic leak occurred in five (n = 5, 16.6%) patients in the handsewn group and eight (n = 7, 13.4%) patients in the circular stapler group. Pyloric manipulation had no significant effect over the leakage rate. Routine upper-gastrointestinal (GI) series done on the fifth or sixth postoperative day did not reveal any of the leaks. Cervical esophagogastric anastomosis using an end-to-side circular stapler is feasible and safe, and has comparable outcomes to handsewn anastomosis in regard of leakage rates or other major surgical or general complications. Postoperative GI series seems to be a poor diagnostic tool for anastomotic leakage and could be omitted as a routine study for occult anastomotic leak.

Defective ubiquitinylation of EGFR mutants of lung cancer confers prolonged signaling.

Several distinct mutations within the kinase domain of the epidermal growth factor receptor (EGFR) are associated with non-small cell lung cancer, but mechanisms underlying their oncogenic potential are incompletely understood. Although normally ligand-induced kinase activation targets EGFR to Cbl-mediated receptor ubiquitinylation and subsequent degradation in lysosomes, we report that certain EGFR mutants escape this regulation. Defective endocytosis characterizes a deletion mutant of EGFR, as well as a point mutant (L858R-EGFR), whose association with c-Cbl and ubiquitinylation are impaired. Our data raise the possibility that refractoriness of L858R-EGFR to downregulation is due to enhanced heterodimerization with the oncogene product HER2, which leads to persistent stimulation.

Viral infections and phenotypic heterogeneity in Gaucher disease.

Gaucher disease, the most common lysosomal storage disorder, is remarkable for its tremendous phenotypic heterogeneity even among patients with the same genotype. Beyond mutations at the 1q21 locus, there may be other genetic and environmental factors that impact on the natural course of Gaucher disease and indeed may trigger symptoms and signs. Among candidate events are viral infections such as the Epstein-Barr virus (EBV) or cytomegalovirus (CMV). The purpose of this study was to ascertain if indeed prior infection with EBV or CMV in patients homozygous for the most common mutation, N370S (1226G), is predictive of a more severe phenotype. Evidence for an EBV virus was IgG and IgM antibodies to early antigen and IgG anti-EBNA. For CMV infection, IgG and IgM antibodies were sought. This study failed to demonstrate any correlation between prior EBV or CMV infection and clinical course of Gaucher disease in patients homozygous for the N370S (1226G) mutation. The only positive finding was a higher level of anti-EBNA antibodies among patients with moderate/severe disease. In conclusion, other than a small subset of patients who showed a pattern comparable to immunosuppression, there was no association between severity of Gaucher disease and prior EBV or CMV infection.

Glutamate 404 is involved in the substrate discrimination of GLT-1, a (Na+ + K+)-coupled glutamate transporter from rat brain.

Sodium-coupled glutamate transporters, located in the plasma membrane of nerve terminals and glial processes, serve to keep its extracellular glutamate concentration below extracellular levels. Moreover, they help in conjunction with diffusion to terminate the transmitter's action in synaptic transmission. We have investigated the role of negatively charged amino acid residues of GLT-1, a cloned (Na+ + K+)-coupled glutamate transporter from rat brain. Using site-directed mutagenesis we modified these negative residues, which are located in hydrophobic surroundings and are highly conserved within the glutamate transporter family. Out of five residues meeting these criteria, three, aspartate 398, glutamate 404, and aspartate 470, are critical for heterologously expressed glutamate transport. This defective transport cannot be attributed to the mere requirement of a negative charge at these positions. After prelabeling of the proteins with [35S]methionine, immunoprecipitation of all mutant transporters indicates that their expression levels are similar to that of wild type. No cryptic activity was revealed by reconstitution experiments aimed to monitor the activity of transporter molecules not located in the plasma membrane. Significantly, whereas all of the mutants at the glutamate 404 position exhibit impaired transport of glutamate, they possess considerable transport of D- and L-aspartate, up to 80% of wild type values. Binding of glutamate is not impaired in these mutants. Our observations indicate that the glutamate 404 residue may be located in the vicinity of the glutamate-aspartate permeation pathway.

Histidine 326 is critical for the function of GLT-1, a (Na+ + K+)-coupled glutamate transporter from rat brain.

Removal of glutamate from the synaptic cleft is carried out by transporter molecules located in presynaptic nerve terminals and fine glial processes surrounding the cleft. Three such transporters, which are approximately 55% identical to each other, have recently been cloned. They catalyze electrogenic transport of this neurotransmitter, which is coupled to the fluxes of three ions: sodium, potassium, and protons (or hydroxyl). One of these transporters, GLT-1, contains 573 amino acids and 6-10 putative membrane-spanning alpha-helices. These helices contain only two positively charged amino acid residues (lysine 298 and histidine 326) that are fully conserved in the glutamate transporters and two related neutral amino acid transporters. Using site-directed mutagenesis we have investigated the role of these residues, each of which was replaced by small hydrophilic as well as by positively charged amino acids. Expression of all replacement mutants at the histidine 326 position reveals that they are severely impaired in sodium-dependent glutamate transport. On the other hand, mutations at lysine 298 retain significant activity, especially if a positively charged amino acid replaces the lysine. After prelabeling of the proteins with [35S]methionine, immunoprecipitation of all mutant transporters indicates that their expression levels are similar to those of wild type. Reconstitution experiments, aimed to reveal the activity of transporter molecules not located in the plasma membrane, indicate that the lowered activity of the K298T and K298N transporters in intact cells is partly due to a targeting defect. Histidine residue 326 appears to be required for the intrinsic activity of the transporter. As histidine residues have been implicated in the mechanism of H+ transport in several systems, we propose that histidine 326 may be involved in the proton translocation accompanying sodium- and potassium-coupled glutamate transport.

Cloning and expression of a rat brain L-glutamate transporter.

Synaptic transmission of most vertebrate synapses is thought to be terminated by rapid transport of the neurotransmitter into presynaptic nerve terminals or neuroglia. L-Glutamate is the major excitatory transmitter in brain and its transport represents the mechanism by which it is removed from the synaptic cleft and kept below toxic levels. Here we use an antibody against a glial L-glutamate transporter from rat brain to isolate a complementary DNA clone encoding this transporter. Expression of this cDNA in transfected HeLa cells indicates that L-glutamate accumulation requires external sodium and internal potassium and transport shows the expected stereospecificity. The cDNA sequence predicts a protein of 573 amino acids with 8-9 putative transmembrane alpha-helices. Database searches indicate that this protein is not homologous to any identified protein of mammalian origin, including the recently described superfamily of neurotransmitter transporters. This protein therefore seems to be a member of a new family of transport molecules.

Counterflow of L-glutamate in plasma membrane vesicles and reconstituted preparations from rat brain.

Membrane vesicles from rat brain exhibit sodium-dependent uptake of L-[3H]glutamate in the absence of any transmembrane ion gradients. The substrate specificity of the process is identical with (Na+ + K+)-coupled L-glutamate accumulation. Although these vesicles are prepared after osmotic shock and are washed repeatedly, they contain about 1.5 nmol/mg of protein endogenous L-glutamate, apparently located inside the vesicles. The affinity of the process (Km approximately 1 microM) is similar to that of (Na+ + K+)-dependent accumulation by the L-glutamate transporter. Membrane vesicles have been disrupted by the detergent cholate, and the solubilized proteins have been subsequently reconstituted into liposomes. The reconstituted proteoliposomes also exhibit the above uptake--with the same characteristics--provided they contain entrapped cold L-glutamate. Counterflow is optimal when sodium is present on both sides of the membrane, but partial activity is still observed when sodium is present either on the inside or on the outside. Increasing the L-glutamate concentration above the Km results in counterflow completely independent of cis sodium. The initial rate of counterflow is 100-200-fold lower than that of net trans potassium dependent flux. The rate of net flux in the presence of trans sodium or lithium is about 10-fold lower than when choline or Tris are used instead. However, the rate of counterflow (no internal potassium present) was not stimulated by replacing internal sodium or lithium by internal choline. Therefore, optimal functioning of the transporter requires internal potassium while internal sodium and lithium are inhibitory.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and reconstitution of the sodium- and potassium-coupled glutamate transport glycoprotein from rat brain.

The sodium- and potassium-coupled L-glutamate transporter from rat brain has been purified to near homogeneity by reconstitution of transport as an assay, assuming that inactivated and active transporters cochromatograph. The purification steps involve lectin chromatography of the membrane proteins solubilized with 3-[(3-chloramidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), fractionation on hydroxylapatite, and ion-exchange chromatography. The specific activity is increased 30-fold. The actual purification is higher since 3-5-fold inactivation occurs during the purification. The efficiency of reconstitution was about 20%. The properties of the pure transporter are fully preserved. They include ion dependence, electrogenicity, affinity, substrate specificity, and stereospecificity. Sodium dodecyl sulfate-polyacrylamide electrophoresis revealed one main band with an apparent molecular mass of around 80 kDa and a few minor bands. Comparison of polypeptide composition with L-glutamate transport activity throughout the fractionation procedure reveals that only the 80-kDa band can be correlated with activity. The GABA transporter, which has the same apparent molecular mass (Radian et al., 1986), is separated from it during the last two purification steps. Immunoblot experiments reveal that the antibodies against the GABA transporter only reacted with fractions exhibiting GABA transport activity and not with those containing the glutamate transporter. We conclude that the 80-kDa band represents the functional sodium- and potassium-coupled L-glutamate transporter.

Enzyme immobilization via monoclonal antibodies I. Preparation of a highly active immobilized carboxypeptidase A.

A novel method for the preparation of highly active immobilized enzymes is described. It is based on the binding of enzymes to suitable carriers via monoclonal antibodies, which bind to the enzyme with high affinity without affecting its catalytic activity. The applicability of the method forwarded has been illustrated by the preparation of two samples of highly active immobilized carboxypeptidase A (CPA) preparations as follows: A mouse monoclonal antibody (mAb 100)to CPA that binds to the enzyme with a high-affinity constant without affecting its catalytic activity was prepared, purified, and characterized. Covalent binding of this monoclonal antibody to Eupergit C (EC) or noncovalent binding to Sepharose-protein A (SPA)yielded the conjugated carriers EC-mAb and SPA.mAb, respectively, which reacted specifically with CPA to give the immobilized enzyme preparations EC-mAb.CPA and SPA.mAb.CPA displaying full catalytic activity and improved stability. At pH 7.5 and a temperature range of 4-37 degrees C an apparent binding constant of approximately 10(8)M(-1) characterizing the interaction of CPA with EC-mAb and SPA.mAb, was obtained. To compare the properties of EC-mAb.CPA and SPA.mAb.CPA with those of immobilized CPA preparations obtained by some representative techniques of covalent binding of the enzyme with a corresponding carrier, the following immobilized CPA preparations were obtained and their properties investigated: EC-CPA (I), a preparation obtained by direct binding of EC with CPA; EC-NH-GA-CPA (II), a derivative obtained by covalent binding of CPA to aminated EC via glutaraldehyde; EC-NH-Su-CPA (III), a CPA derivative obtained by binding the enzyme to aminated EC via a succinyl residue; and EC-HMD-GA-CPA (IV), obtained by binding the enzyme via glutaraldehyde to a hexamethylene diamine derivative of EC. Full enzymic activity for all of the bound enzyme, such as that recorded for the immobilized CPA preparations EC-mAb.CPA and SPA.mAb.CPA, was not detected in any of the insoluble covalently bound enzyme preparations.

Interaction of carboxypeptidase A with monoclonal antibodies.

Several mouse monoclonal antibodies to carboxypeptidase A (CPA) were prepared and purified, and their interaction with the enzyme was investigated. CPA is a well-characterized zinc-containing exopeptidase exhibiting peptidase as well as esterase activity. The antibodies obtained could be classified as follows: antibodies inhibiting mainly the peptidase activity of the enzyme, antibodies inhibiting mainly its esterase activity, antibodies affecting both activities, and antibodies which bind to the enzyme but have no marked effect on its catalytic properties. Binding constants of approximately 10(6) M-1 were obtained for most of the antibody-enzyme complexes tested. Additional information on the effect of the monoclonal antibodies on the active site of CPA was obtained by determining the change in the circular dichroism spectra of arsanilazotyrosine-248 carboxypeptidase A occurring as a result of the interaction of the enzyme with the antibodies studied. These findings suggest that CPA possesses at least three different specific antigenic sites, and that the active site of the enzyme for its peptidase activity differs from that for its esterase activity, though both sites seem to overlap to a considerable extent.