A Novel Binding Interaction For The Paxillin Ld3 Motif: Paxillin Ld3 Mediates Merlin-Paxillin Binding At Paxillin Binding Domain 1

Neurofibromatosis type 2, an autosomal dominant genetic disorder, causes predisposed individuals to develop various benign central and peripheral nervous system tumors. The characteristic tumors of this disease are schwannomas, which are tumors of the Schwann cells, typically on the vestibular nerve. These and the other associated tumors slowly compress nervous system structures causing deafness and loss of balance, resulting in an average life-span of less than 40 years. The product of the Nf2 gene is the protein named merlin or schwannomin. In individuals diagnosed with NF2, merlin is either absent or mutated to the point of inactivation. As such, merlin functions as a negative growth regulator in that it suppresses tumor growth. Being that NF2 is predominately a disease of the Schwann cells, merlin\u27s functional role within the signal transduction pathways of Schwann cell growth and differentiation are being investigated. This thesis explores the molecular relationships between merlin and its various interactors within Schwann cells, and illuminates one step in elucidating merlin\u27s functional mechanism of action. Merlin has been shown to associate with paxillin in a density-dependant manner and to bind directly to paxillin through two specific paxillin binding domains. Individual paxillin LD domain fusion proteins were produced, as well as recombinant merlin lacking the paxillin binding domains. Direct binding assays were performed in order to determine which specific paxillin domains merlin might interact with directly. The results indicate that, in vitro, merlin binds, through its PBD1 domain, to the paxillin LD3 motif. Supporting this data, the results also demonstrate that when the merlin PBD1 domain is deleted, merlin binding to paxillin LD3 is abrogated. The direct binding shown here between paxillin and merlin, coupled with research demonstrating that merlin is present in β1 integrin immuno-precipitations, leads to the question of whether merlin binds directly to β1 integrin or associates with β1 integrin through paxillin. Using direct binding assays, this research shows that the merlin C-terminus binds directly to the cytoplasmic domain of β1 integrin, in vitro. Lastly, since merlin is an ERM family protein and has been shown to dimerize with ezrin (another ERM family member), and because merlin has been shown to bind directly to paxillin, the question asked is whether paxillin can interact directly with ezrin. The results indicate that paxillin can bind directly to the N-terminus of ezrin, in vitro. The findings presented here, when examined together, provide a framework for the proposal of a model in which paxillin LD3 mediates the localization of merlin to the plasma membrane, where it associates with the β1 integrin cytoplasmic domain and ezrin. These results and the proposed model offer additional insight into the mechanism of action of merlin\u27s negative growth regulating function in Schwann cells

AI is a viable alternative to high throughput screening: a 318-target study

: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery

Formation Of A Β1 Integrin Signaling Complex In Schwann Cells Is Independent Of Rho

Schwann cell adhesion to basal lamina is essential for peripheral nerve development. β1 integrin receptors for extracellular matrix cooperate with other receptors to transmit signals that coordinate cell cycle progression and initiation of differentiation, including myelin-specific gene expression. In Schwann cell/sensory neuron cocultures, β1 integrins complex with focal adhesion kinase (FAK), fyn kinase, paxillin, and schwannomin in response to basal lamina adhesion. To study the assembly of this signaling complex in Schwann cells (SCs), we induced β1 integrin clustering on suspended cells using an immobilized antibody and recovered a complex containing β1 integrin, FAK, paxillin, and schwannomin. In adherent subconfluent cells, the proteins colocalized to filopodia, ruffling membranes and focal contacts. We assessed the role of rhoGTPase in the process of integrin complex assembly by introducing C3 transferase (C3T), a rho inhibitor, into the cells. Although C3T caused dose-dependent morphological abnormalities, FAK, paxillin, and schwannomin were able to coimmunoprecipitate with β1 integrin. Additionally, colocalization of FAK, paxillin, and schwannomin with β1 integrin in filopodia and small focal contacts remained unchanged. We conclude that SCs do not require active rho to recruit signaling and structural proteins to β1 integrins clustered at the plasma membrane. Rho is required to establish large focal adhesions and to spread and stabilize plasma membrane extensions. © 2003 Wiley-Liss, Inc

Lipopolyamine Treatment Increases The Efficacy Of Intoxication With Saporin And An Anticancer Saporin Conjugate

Saporin is a type I ribosome-inactivating protein that is often appended with a cell-binding domain to specifically target and kill cancer cells. Urokinase plasminogen activator (uPA)-saporin, for example, is an anticancer toxin that consists of a chemical conjugate between the human uPA and native saporin. Both saporin and uPA-saporin enter the target cell by endocytosis and must then escape the endomembrane system to reach the cytosolic ribosomes. The latter process may represent a rate-limiting step for intoxication and would therefore directly affect toxin potency. In the present study, we document two treatments (shock with dimethylsulfoxide and lipopolyamine coadministration) that generate substantial cellular sensitization to saporin/uPA-saporin. With the use of lysosome-endosome X (LEX)1 and LEX2 mutant cell lines, an endosomal trafficking step preceding cargo delivery to the late endosomes was identified as a major site for the dimethylsulfoxide-facilitated entry of saporin into the cytosol. Dimethylsulfoxide and lipopolyamines are known to disrupt the integrity of endosome membranes, so these reagents could facilitate the rapid movement of toxin from permeabilized endosomes to the cytosol. However, the same pattern of toxin sensitization was not observed for dimethylsulfoxide- or lipopolyamine-treated cells exposed to diphtheria toxin, ricin, or the catalytic A chain of ricin. The sensitization effects were thus specific for saporin, suggesting a novel mechanism of saporin translocation by endosome disruption. Lipopolyamines have been developed as in vivo gene therapy vectors; thus, lipopolyamine coadministration with uPA-saporin or other saporin conjugates could represent a new approach for anticancer toxin treatments. © 2007 The Authors