Tamara C. Petrucci

Diversity and Functional Consequences of Germline and Somatic PTPN11 Mutations in Human Disease

Germline mutations in PTPN11, the gene encoding the protein tyrosine phosphatase SHP-2, cause Noonan syndrome (NS) and the clinically related LEOPARD syndrome (LS), whereas somatic mutations in the same gene contribute to leukemogenesis. On the basis of our previously gathered genetic and biochemical data, we proposed a model that splits NS- and leukemia-associated PTPN11 mutations into two major classes of activating lesions with differential perturbing effects on development and hematopoiesis. To test this model, we investigated further the diversity of germline and somatic PTPN11 mutations, delineated the association of those mutations with disease, characterized biochemically a panel of mutant SHP-2 proteins recurring in NS, LS, and leukemia, and performed molecular dynamics simulations to determine the structural effects of selected mutations. Our results document a strict correlation between the identity of the lesion and disease and demonstrate that NS-causative mutations have less potency for promoting SHP-2 gain of function than do leukemia-associated ones. Furthermore, we show that the recurrent LS-causing Y279C and T468M amino acid substitutions engender loss of SHP-2 catalytic activity, identifying a previously unrecognized behavior for this class of missense PTPN11 mutations.

Evidence for in Situ and in VitroAssociation between β-Dystroglycan and the Subsynaptic 43K Rapsyn Protein CONSEQUENCE FOR ACETYLCHOLINE RECEPTOR CLUSTERING AT THE SYNAPSE

The accumulation of dystrophin and associated proteins at the postsynaptic membrane of the neuromuscular junction and their co-distribution with nicotinic acetylcholine receptor (AChR) clusters in vitro suggested a role for the dystrophin complex in synaptogenesis. Co-transfection experiments in which α- and β-dystroglycan form a complex with AChR and rapsyn, a peripheral protein required for AChR clustering (Apel, D. A., Roberds, S. L., Campbell, K. P., and Merlie, J. P. (1995)Neuron 15, 115–126), suggested that rapsyn functions as a link between AChR and the dystrophin complex. We have investigated the interaction between rapsyn and β-dystroglycan in TorpedoAChR-rich membranes using in situ and in vitroapproaches. Cross-linking experiments were carried out to study the topography of postsynaptic membrane polypeptides. A cross-linked product of 90 kDa was labeled by antibodies to rapsyn and β-dystroglycan; this demonstrates that these polypeptides are in close proximity to one another. Affinity chromatography experiments and ligand blot assays using rapsyn solubilized from TorpedoAChR-rich membranes and constructs containing β-dystroglycan C-terminal fragments show that a rapsyn-binding site is present in the juxtamembranous region of the cytoplasmic tail of β-dystroglycan. These data point out that rapsyn and dystroglycan interact in the postsynaptic membrane and thus reinforce the notion that dystroglycan could be involved in synaptogenesis.

Dystroglycan distribution in adult mouse brain : A light and electron microscopy study

Dystroglycan, originally identified in muscle as a component of the dystrophin-associated glycoprotein complex, is a ubiquitously expressed cell-surface receptor that forms a transmembrane link between the extracellular matrix and the cytoskeleton. It contains two subunits, alpha and beta, formed by proteolytic cleavage of a common precursor. In the brain, different neuronal subtypes and glial cells may express dystroglycan in complex with distinct cytoplasmic proteins such as dystrophin, utrophin and their truncated forms. To examine the distribution of dystroglycan in adult mouse brain, we raised antibodies against the recombinant amino- and carboxyl-terminal domains of alpha-dystroglycan. On western blot, the antibodies recognized specifically alpha-dystroglycan in cerebellar extracts. Using light microscopy, alpha-dystroglycan was found in neurons of the cerebral cortex, hippocampus, olfactory bulb, basal ganglia, thalamus, hypothalamus, brainstem and cerebellum, where dystrophin and its truncated isoforms are also known to be present. Electron microscopy revealed that alpha-dystroglycan immunoreactivity was preferentially associated with the postsynaptic specializations. Dystroglycan immunostaining was also detected in perivascular astrocytes and in those facing the pia mater, where utrophin and dystrophin truncated isoforms are present. The cell body and endfeet of astrocytes around blood vessels and the endothelial cells at the blood-brain barrier also expressed dystroglycan. From these data, we suggest that dystroglycan, by bridging the extracellular matrix and the cytoskeleton, may play an important functional role at specialized intercellular contacts, synapses and the blood-brain barrier, whose structural and functional organization strictly depend on the integrity of the extracellular matrix-cytoskeleton linkage.

Wheat protein inhibitors of α-amylase

Abstract The location in the seed, molecular properties and biological role of protein α-amylase inhibitors from wheat are discussed. Inhibition specificity of albumin inhibitors and structural features essential for interaction with inhibited amylases are also examined. The possible significance of these naturally occurring inhibitors in relation to their presence in foods in active form is described. Finally, genetic aspects of the albumin inhibitor production and the possibility of improving nutritional value and insect re

Association of the dystroglycan complex isolated from bovine brain synaptosomes with proteins involved in signal transduction.

Abstract: Dystroglycan is a transmembrane heterodimeric complex of α and β subunits that links the extracellular matrix to the cell cytoskeleton. It was originally identified in skeletal muscle, where it anchors dystrophin to the sarcolemma. Dystroglycan is also highly expressed in nonmuscle tissues, including brain. To investigate the molecular interactions of dystroglycan in the CNS, we fractionated a digitonin‐soluble extract from bovine brain synaptosomes by laminin‐affinity chromatography and characterized the protein components. The 120‐kDa α‐dystroglycan was the major 125I‐laminin‐labeled protein detected by overlay assay. This complex, in addition to β‐dystroglycan, was also found to contain Grb2 and focal adhesion kinase p125FAK (FAK). Anti‐FAK antibodies co‐immunoprecipitated Grb2 with FAK. However, no direct interaction between β‐dystroglycan and FAK was detected by co‐precipitation assay. Grb2, an adaptor protein involved in signal transduction and cytoskeleton organization, has been shown to bind β‐dystroglycan. We isolated both FAK and Grb2 from synaptosomal extracts by chromatography on immobilized recombinant β‐dystroglycan. In the CNS, FAK phosphorylation has been linked to membrane depolarization and neurotransmitter receptor activation. At the synapses, the adaptor protein Grb2 may mediate FAK‐β‐dystroglycan interaction, and it may play a role in transferring information between the dystroglycan complex and other signaling pathways.

Peroxynitrite Induces Tyrosine Nitration and Modulates Tyrosine Phosphorylation of Synaptic Proteins

Abstract : Peroxynitrite, the product of the radical‐radical reaction between nitric oxide and superoxide anion, is a potent oxidant involved in tissue damage in neurodegenerative disorders. We investigated the modifications induced by peroxynitrite in tyrosine residues of proteins from synaptosomes. Peroxynitrite treatment (≥50 μM) induced tyrosine nitration and increased tyrosine phosphorylation. Synaptophysin was identified as one of the major nitrated proteins and pp60src kinase as one of the major phosphorylated substrates. Further fractionation of synaptosomes revealed nitrated synaptophysin in the synaptic vesicles, whereas phosphorylated pp60src was enriched in the postsynaptic density fraction. Tyrosine phosphorylation was increased by treatment with 50‐500 μM peroxynitrite and decreased by higher concentrations, suggesting a possible activation/inactivation of kinases. Immunocomplex kinase assay proved that peroxynitrite treatment of synaptosomes modulated the pp60src autophosphorylation activity. The addition of bicarbonate (CO2 1.3 mM) produced a moderate enhancing effect on some nitrate proteins but significantly protected the activity of pp60src against peroxynitrite‐mediated inhibition so that at 1 mM peroxynitrite, the kinase was still more active than in untreated synaptosomes. The phosphotyrosine phosphatase activity of synaptosomes was inhibited by peroxynitrite (≥50 μM) but significantly protected by CO2. Thus, the increase of phosphorylation cannot be attributed to peroxynitrite‐mediated inhibition of phosphatases. We suggest that peroxynitrite may regulate the posttranslational modification of tyrosine residues in pre‐ and postsynaptic proteins. Identification of the major protein targets gives insight into the pathways possibly involved in neuronal degeneration associated with peroxynitrite overproduction.

Anomalous dystroglycan in carcinoma cell lines

Dystroglycan is a receptor responsible for crucial interactions between extracellular matrix and cytoplasmic space. We provide the first evidence that dystroglycan is truncated. In HC11 normal murine and the 184B5 non‐tumorigenic mammary human cell lines, the expected β‐dystroglycan 43 kDa band was found but human breast T47D, BT549, MCF7, colon HT29, HCT116, SW620, prostate DU145 and cervical HeLa cancer cells expressed an anomalous ≈31 kDa β‐dystroglycan band. α‐Dystroglycan was udetectable in most of the cell lines in which β‐dystroglycan was found as a ≈31 kDa species. An anomalous ≈31 kDa β‐dystroglycan band was also observed in N‐methyl‐N‐nitrosurea‐induced primary rat mammary tumours. Reverse transcriptase polymerase chain reaction experiments confirmed the absence of alternative splicing events and/or expression of eventual dystroglycan isoforms. Using protein extraction procedures at low‐ and high‐ionic strength, we demonstrated that both the 43 kDa and ≈31 kDa β‐dystroglycan bands harbour their transmembrane segment.

Comparison of wheat albumin inhibitors of α-amylase and trypsin

Abstract Wheat albumins were extracted from whole wheat flour with 150 mM sodium chloride solution and precipitated between 0·4 and 1·8 M ammonium sulphate. The albumin precipitate was separated by gel filtration on Sephadex G100 into five peaks. Three peaks (II, III, and IV), whose MWs were 60 000, 24 000 and 12 500 daltons respectively, were active toward several insect α-amylases, whereas only peak III inhibited human saliva and pancreatic α-amylases. Peaks III and IV also inhibited trypsin. In each active peak, we found several α-amylase inhibitors slightly different in their electrophoretic mobilities in a Tris—glycine buffer system (pH 8·5), whereas only one major trypsin inhibitor was present in peaks III and IV. In contrast to α-amylase inhibitors that were all anodic, trypsin inhibitors migrated to the cathode under our experimental conditions. From a quantitative standpoint, wheat albumins that inhibit trypsin are negligible, whereas about 2/3 of the total albumin inhibits amylases from different origins. All inhibitor components of peak III were active toward both insect and mammalian α-amylases. Moreover, they reversibly dissociated in the presence of 6 M guanidine hydrochloride giving two similar subunits.

Protein phosphatase inhibitors induce modification of synapse structure and tau hyperphosphorylation in cultured rat hippocampal neurons

Protein phosphatase inhibitors, okadaic acid and Caliculin A, were used to investigate how perturbation of phosphorylation and dephosphorylation processes might affect neurite and synapse structure in cultures of fetal rat hippocampal neurons. Drug treatments induced neuritic tree modification, with retraction of the processes and the appearance of dilatations along the neurites. The characteristic dotlike pattern of immunoreactivity of synaptic vesicle proteins disappeared. Normal synapses were extremely rare by ultrastructural observation. Vesicles of various diameters accumulated in the dilatations, as did organelles and amorphous material, suggesting impaired axonal transport. Hyperphosphorylation of tau protein was also observed as indicated by the shift in the electrophoretic mobility of a 32P‐labeled 55‐kDa band and by immunoblot with epitope‐specific tau antibody.

Peroxynitrite affects exocytosis and SNARE complex formation and induces tyrosine nitration of synaptic proteins

The reactive species peroxynitrite, formed via the near diffusion‐limited reaction of nitric oxide and superoxide anion, is a potent oxidant that contributes to tissue damage in neurodegenerative disorders. Peroxynitrite readily nitrates tyrosine residues in proteins, producing a permanent modification that can be immunologically detected. We have previously demonstrated that in the nerve terminal, nitrotyrosine immunoreactivity is primarily associated with synaptophysin. Here we identify two other presynaptic proteins nitrated by peroxynitrite, Munc‐18 and SNAP25, both of which are involved in sequential steps leading to vesicle exocytosis. To investigate whether peroxynitrite affects vesicle exocytosis, we used the fluorescent dye FM1‐43 to label a recycling population of secretory vesicles within the synaptosomes. Bolus addition of peroxynitrite stimulated exocytosis and glutamate release. Notably, these effects were strongly reduced in the presence of NaHCO3, indicating that peroxynitrite acts mainly intracellularly. Furthermore, peroxynitrite enhanced the formation of the sodium dodecyl sulfate‐resistant SNARE complex in a dose‐dependent manner (100–1000 µm) and induced the formation of 3‐nitrotyrosine in proteins of SNARE complex. These data suggest that modification(s) of synaptic vesicle proteins induced by peroxynitrite may affect protein–protein interactions in the docking/fusion steps, thus promoting exocytosis, and that, under excessive production of superoxide and nitric oxide, neurons may up‐regulate neuronal signaling.