Structure-guided design fine-tunes pharmacokinetics, tolerability, and antitumor profile of multispecific frizzled antibodies.

Aberrant activation of Wnt/ß-catenin signaling occurs frequently in cancer. However, therapeutic targeting of this pathway is complicated by the role of Wnt in stem cell maintenance and tissue homeostasis. Here, we evaluated antibodies blocking 6 of the 10 human Wnt/Frizzled (FZD) receptors as potential therapeutics. Crystal structures revealed a common binding site for these monoclonal antibodies (mAbs) on FZD, blocking the interaction with the Wnt palmitoleic acid moiety. However, these mAbs displayed gastrointestinal toxicity or poor plasma exposure in vivo. Structure-guided engineering was used to refine the binding of each mAb for FZD receptors, resulting in antibody variants with improved in vivo tolerability and developability. Importantly, the lead variant mAb significantly inhibited tumor growth in the HPAF-II pancreatic tumor xenograft model. Taken together, our data demonstrate that anti-FZD cancer therapeutic antibodies with broad specificity can be fine-tuned to navigate in vivo exposure and tolerability while driving therapeutic efficacy.

Next-generation sequencing identifies rare variants associated with Noonan syndrome.

Noonan syndrome (NS) is a relatively common genetic disorder, characterized by typical facies, short stature, developmental delay, and cardiac abnormalities. Known causative genes account for 70-80% of clinically diagnosed NS patients, but the genetic basis for the remaining 20-30% of cases is unknown. We performed next-generation sequencing on germ-line DNA from 27 NS patients lacking a mutation in the known NS genes. We identified gain-of-function alleles in Ras-like without CAAX 1 (RIT1) and mitogen-activated protein kinase kinase 1 (MAP2K1) and previously unseen loss-of-function variants in RAS p21 protein activator 2 (RASA2) that are likely to cause NS in these patients. Expression of the mutant RASA2, MAP2K1, or RIT1 alleles in heterologous cells increased RAS-ERK pathway activation, supporting a causative role in NS pathogenesis. Two patients had more than one disease-associated variant. Moreover, the diagnosis of an individual initially thought to have NS was revised to neurofibromatosis type 1 based on an NF1 nonsense mutation detected in this patient. Another patient harbored a missense mutation in NF1 that resulted in decreased protein stability and impaired ability to suppress RAS-ERK activation; however, this patient continues to exhibit a NS-like phenotype. In addition, a nonsense mutation in RPS6KA3 was found in one patient initially diagnosed with NS whose diagnosis was later revised to Coffin-Lowry syndrome. Finally, we identified other potential candidates for new NS genes, as well as potential carrier alleles for unrelated syndromes. Taken together, our data suggest that next-generation sequencing can provide a useful adjunct to RASopathy diagnosis and emphasize that the standard clinical categories for RASopathies might not be adequate to describe all patients.

Global proteomic assessment of the classical protein-tyrosine phosphatome and "Redoxome".

Protein-tyrosine phosphatases (PTPs), along with protein-tyrosine kinases, play key roles in cellular signaling. All Class I PTPs contain an essential active site cysteinyl residue, which executes a nucleophilic attack on substrate phosphotyrosyl residues. The high reactivity of the catalytic cysteine also predisposes PTPs to oxidation by reactive oxygen species, such as H(2)O(2). Reversible PTP oxidation is emerging as an important cellular regulatory mechanism and might contribute to diseases such as cancer. We exploited these unique features of PTP enzymology to develop proteomic methods, broadly applicable to cell and tissue samples, that enable the comprehensive identification and quantification of expressed classical PTPs (PTPome) and the oxidized subset of the PTPome (oxPTPome). We find that mouse and human cells and tissues, including cancer cells, display distinctive PTPomes and oxPTPomes, revealing additional levels of complexity in the regulation of protein-tyrosine phosphorylation in normal and malignant cells.

SHP2 tyrosine phosphatase converts parafibromin/Cdc73 from a tumor suppressor to an oncogenic driver.

Deregulation of SHP2 is associated with malignant diseases as well as developmental disorders. Although SHP2 is required for full activation of RAS signaling, other potential roles in cell physiology have not been elucidated. Here we show that SHP2 dephosphorylates parafibromin/Cdc73, a core component of the RNA polymerase II-associated factor (PAF) complex. Parafibromin is known to act as a tumor suppressor that inhibits cyclin D1 and c-myc by recruiting SUV39H1 histone methyltransferase. However, parafibromin can also act in the opposing direction by binding ß-catenin, thereby activating promitogenic/oncogenic Wnt signaling. We found that, on tyrosine dephosphorylation by SHP2, parafibromin acquires the ability to stably bind ß-catenin. The parafibromin/ß-catenin interaction overrides parafibromin/SUV39H1-mediated transrepression and induces expression of Wnt target genes, including cyclin D1 and c-myc. Hence, SHP2 governs the opposing functions of parafibromin, deregulation of which may cause the development of tumors or developmental malformations.

Phosphotyrosine mapping in Bcr/Abl oncoprotein using phosphotyrosine-specific immonium ion scanning.

Bcr/Abl is a fusion oncoprotein that is of paramount importance in chronic myelogenous leukemia and acute lymphocytic leukemia. The tyrosine-phosphorylated fraction of the p185 form of Bcr/Abl was isolated by immunoprecipitation with an anti-phosphotyrosine antibody and SDS-PAGE. The tryptic digest of the gel-separated protein was prefractionated on POROS R2/OLIGO R3 microcolumns and subjected to phosphotyrosine mapping by precursor ion scanning in positive ion mode utilizing the immonium ion of phosphotyrosine, also called phosphotyrosine-specific immonium ion scanning, on a quadrupole time-of-flight tandem mass spectrometer. In total, nine different phosphorylated tyrosine residues were unambiguously localized in 12 different precursor ions. These phosphorylation sites correspond to three previously described phosphotyrosine residues and six novel tyrosine phosphorylation sites, and most of them were not predicted by the phosphorylation motif prediction programs ProSite, NetPhos, or ScanSite. This study shows the power of phosphotyrosine-specific immonium ion scanning for sensitive phosphotyrosine mapping when limited amounts of samples are available.

Cloning of a novel phosphotyrosine binding domain containing molecule, Odin, involved in signaling by receptor tyrosine kinases.

We have used a proteomic approach using mass spectrometry to identify signaling molecules involved in receptor tyrosine kinase signaling pathways. Using affinity purification by anti-phosphotyrosine antibodies to enrich for tyrosine phosphorylated proteins, we have identified a novel signaling molecule in the epidermal growth factor receptor signaling pathway. This molecule, designated Odin, contains several ankyrin repeats, two sterile alpha motifs and a phosphotyrosine binding domain and is ubiquitously expressed. Using antibodies against endogenous Odin, we show that it undergoes tyrosine phosphorylation upon addition of growth factors such as EGF or PDGF but not by cytokines such as IL-3 or erythropoietin. Immunofluorescence experiments as well as Western blot analysis on subcellular fractions demonstrated that Odin is localized to the cytoplasm both before and after growth factor treatment. Deletion analysis showed that the phosphotyrosine binding domain of Odin is not required for its tyrosine phosphorylation. Overexpression of Odin, but not an unrelated adapter protein, Grb2, inhibited EGF-induced activation of c-Fos promoter. Microinjection of wild-type or a mutant version lacking the PTB domain into NIH3T3 fibroblasts inhibited PDGF-induced mitogenesis. Taken together, our results indicate that Odin may play a negative role in growth factor receptor signaling pathways.

Inhibition of adipocyte differentiation by resistin-like molecule alpha. Biochemical characterization of its oligomeric nature.

A novel family of cysteine-rich secreted proteins with unique tissue distribution has recently been identified. One of the members, resistin (for "resistance to insulin"), also called FIZZ3, was identified in a screen for molecules that are down-regulated in mature adipocytes upon administration of thiazolidinediones. The prototypical member of this family was originally identified from bronchoalveolar lavage fluid of inflamed lungs and designated FIZZ1 ("found in inflammatory zone"). This molecule was also found to be highly expressed in adipose tissue and was named resistin-like molecule alpha (RELMalpha). Here we demonstrate that RELMalpha inhibits the differentiation of 3T3-L1 preadipocytes into adipocytes. RELMalpha has no effect on proliferation of 3T3-L1 preadipocytes. Pretreatment of 3T3-L1 preadipocytes with RELMalpha does not affect insulin- or platelet-derived growth factor-induced mitogenesis. IRS-1 phosphorylation and glucose transport stimulated by insulin in mature adipocytes were also unaffected by RELMalpha. We show that RELMalpha forms disulfide-linked homooligomers based on results from electrophoresis under reducing and nonreducing conditions, coimmunoprecipitation experiments as well as by mass spectrometry. In addition, RELMalpha is able to form heterooligomers with resistin but not RELMbeta. Since RELMalpha is expressed by adipose tissue and it is a secreted factor, our findings suggest that RELMalpha may be involved in the control of the adipogenesis as well as in the process of muscle differentiation.

A proteomic approach for identification of secreted proteins during the differentiation of 3T3-L1 preadipocytes to adipocytes.

We have undertaken a systematic proteomic approach to purify and identify secreted factors that are differentially expressed in preadipocytes versus adipocytes. Using one-dimensional gel electrophoresis combined with nanoelectrospray tandem mass spectrometry, proteins that were specifically secreted by 3T3-L1 preadipocytes or adipocytes were identified. In addition to a number of previously reported molecules that are up- or down-regulated during this differentiation process (adipsin, adipocyte complement-related protein 30 kDa, complement C3, and fibronectin), we identified four secreted molecules that have not been shown previously to be expressed differentially during the process of adipogenesis. Pigment epithelium-derived factor, a soluble molecule with potent antiangiogenic properties, was found to be highly secreted by preadipocytes but not adipocytes. Conversely, we found hippocampal cholinergic neurostimulating peptide, neutrophil gelatinase-associated lipocalin, and haptoglobin to be expressed highly by mature adipocytes. We also used liquid chromatography-based separation followed by automated tandem mass spectrometry to identify proteins secreted by mature adipocytes. Several additional secreted proteins including resistin, secreted acidic cysteine-rich glycoprotein/osteonectin, stromal cell-derived factor-1, cystatin C, gelsolin, and matrix metalloprotease-2 were identified by this method. To our knowledge, this is the first study to identify several novel secreted proteins by adipocytes by a proteomic approach using mass spectrometry.

A novel Src homology 2 domain-containing molecule, Src-like adapter protein-2 (SLAP-2), which negatively regulates T cell receptor signaling.

We have cloned a novel adapter protein containing Src homology 2 and Src homology 3 domains similar to the Src family of tyrosine kinases. This molecule lacks a catalytic tyrosine kinase domain and is related to a previously identified protein, Src-like adapter protein (SLAP), and is therefore designated SLAP-2. Northern blot analysis indicates that SLAP-2 is predominantly expressed in the immune system. Jurkat T cells express SLAP-2 protein and overexpression of SLAP-2 in these cells negatively regulates T cell receptor signaling as assessed by interleukin-2 promoter or NF-AT promoter reporter constructs. Mutational analysis revealed that an intact SH2 domain of SLAP-2 is essential for this inhibitory effect, whereas mutation of the SH3 domain alone has no effect. This inhibitory effect is upstream of the activation of Ras and increase of intracellular calcium levels, as no inhibition was observed when the cells were activated by phorbol ester plus ionomycin. SLAP-2 interacts with Cbl in vivo in a phosphorylation independent manner and with ZAP-70 and T cell receptor zeta chain upon T cell receptor activation. Finally, we show that the mutation of a predicted myristoylation site within the NH(2)-terminal of SLAP-2 is essential for its inhibitory effect. This report therefore implicates SLAP and SLAP-2 as a family of adapter proteins that negatively regulate T cell receptor signaling.

Tyrosine phosphorylation mapping of the epidermal growth factor receptor signaling pathway.

Phosphorylation is one of the most common forms of protein modification. The most frequent targets for protein phosphorylation in eukaryotes are serine and threonine residues, although tyrosine residues also undergo phosphorylation. Many of the currently applied methods for the detection and localization of protein phosphorylation sites are mass spectrometry-based and are biased against the analysis of tyrosine-phosphorylated residues because of the stability and low reactivity of phosphotyrosines. To overcome this lack of sensitive methods for the detection of phosphotyrosine-containing peptides, we have recently developed a method that is not affected by the more predominant threonine or serine phosphorylation within cells. It is based on the specific detection of immonium ion of phosphotyrosine at 216.043 Da and does not require prior knowledge of the protein sequence. In this report, we describe the first application of this new method in a proteomic strategy. Using anti-phosphotyrosine antibodies for immunoprecipitation and one-dimensional gel electrophoresis, we have identified 10 proteins in the epidermal growth factor receptor signaling pathway, of which 8 have been shown previously to be involved in epidermal growth factor signaling. Most importantly, in addition to several known tyrosine phosphorylation sites, we have identified five novel sites on SHIP-2, Hrs, Cbl, STAM, and STAM2, most of which were not predicted to be phosphorylated. Because of its sensitivity and selectivity, this approach will be useful in proteomic approaches to study tyrosine phosphorylation in a number of signal transduction pathways.

Variabilidad interobservadores al delimitar regiones de interés en gamagramas computarizados / Interobserver variability in RIO drawing in computerized scan processing

Antecedentes. En la actualidad, de los departamentos de medicina nuclear cuentan con cámaras de centelleo para gamagrafía computarizada. Para el radiorrenograma se adquieren 90 imágenes de 16 segundos cada una, se integran en una sola y sobre ella se trazan regiones de interés (RDI) dibujado el contorno de los riñones y de la vejiga, por medio de una palanca móvil ("joy stick"). El programa utliza la radiactividad de cada RDI para generar curvas de actividad/tiempo, y calcular el tiempo de máxima actividad renal (Tmax) y el tiempo que tarde el riñón en eliminar la mitad de esta radiactividad T½). El dibujo del contorno de órgano, al trazar las RDI, es el único paso en que interviene directamente el operador por lo que cabe la posibilidad de que los resultados dependan de su habilidad y experiencia, y por ende, que hubiera diferencias interoperadores que repercutieran en la interpretación clínica de los gamagramas. Objetivo. Establecer la variabilidad interoperadores de cuatro personas experimentadas en el trazado de RDI en imágenes renales. Material y métodos. Los cuatro operadores trazaron las RDI para obtener la Tmax Y T½ de 38 riñones de 20 pacientes (dos trasplantados). Se calculó el CV interoperadores de Tmax y T½ de los 38 riñones. Resultados. Globalmente hubo mayor variabilidad interobservadores en T½ que en Tmax. Cuatro riñones mostraron pequeñas diferencias interoperadores en Tmax y/o T½ pero suficientes para que discreparan sus clasificaciones de normal/retardado. La separación de los datos en tres grupos (T = Tmax y T½ normales; 2 = Tmax normal y T½ retardada; 3 = ambas retardadas) mostró que había diferencias intergrupos significativas en el CV interobservadores de la Tmax (Kruskal-Wallis p = 0.032) que obedecían a que la variabilidad fue mayor en el grupo 2 (6 de 11 riñones con CV >4 por ciento) que en los grupos 1 y 3 (ninguno con CV >4 por ciento). Conclusiones. 1. Las discrepancias interoperadores tuvieron repercusión sólo en algunos casos en que los parámetros estaban ligeramente retardados (8-9 min en Tmax, 15-20 min en T½). 2. No tenermos una explicación de por qué hubo mayor variabilidad interoperadores en Tmax de los riñones del grupo 2 (Tmax normal con T½ retardada). 3. Creemos que un estudio de la variabilidad interoperador de estas mismas cuatro personas puede aclarar algunas de las observaciones de este estudio