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      • Record: found
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      The Anti-Cancer IgM Monoclonal Antibody PAT-SM6 Binds with High Avidity to the Unfolded Protein Response Regulator GRP78

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          Abstract

          The monoclonal IgM antibody PAT-SM6 derived from human tumours induces apoptosis in tumour cells and is considered a potential anti-cancer agent. A primary target for PAT-SM6 is the unfolded protein response regulator GRP78, over-expressed externally on the cell surface of tumour cells. Small angle X-ray scattering (SAXS) studies of human GRP78 showed a two-domain dumbbell-shaped monomer, while SAXS analysis of PAT-SM6 revealed a saucer-shaped structure accommodating five-fold symmetry, consistent with previous studies of related proteins. Sedimentation velocity analysis of GRP78 and PAT-SM6 mixtures indicated weak complex formation characterized by dissociation constants in the high micromolar concentration range. In contrast, enzyme-linked immunosorbant assays (ELISAs) showed strong and specific interactions between PAT-SM6 and immobilized GRP78. The apparent binding constant estimated from a PAT-SM6 saturation curve correlated strongly with the concentration of GRP78 used to coat the microtiter tray. Experiments using polyclonal antiGRP78 IgG antibodies or a monoclonal IgG derivative of PAT-SM6 did not show a similar dependence. Competition experiments with soluble GRP78 indicated more effective inhibition of PAT-SM6 binding at low GRP78 coating concentrations. These observations suggest an avidity-based binding mechanism that depends on the multi-point attachment of PAT-SM6 to GRP78 clustered on the surface of the tray. Analysis of ELISA data at high GRP78 coating concentrations yielded an apparent dissociation constant of approximately 4 nM. We propose that the biological action of PAT-SM6 in tumour cell apoptosis may depend on the multivalent nature of PAT-SM6 and the high avidity of its interaction with multiple GRP78 molecules clustered on the tumour cell surface.

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          Most cited references 29

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          Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and lamm equation modeling.

           Peter Schuck (2000)
          A new method for the size-distribution analysis of polymers by sedimentation velocity analytical ultracentrifugation is described. It exploits the ability of Lamm equation modeling to discriminate between the spreading of the sedimentation boundary arising from sample heterogeneity and from diffusion. Finite element solutions of the Lamm equation for a large number of discrete noninteracting species are combined with maximum entropy regularization to represent a continuous size-distribution. As in the program CONTIN, the parameter governing the regularization constraint is adjusted by variance analysis to a predefined confidence level. Estimates of the partial specific volume and the frictional ratio of the macromolecules are used to calculate the diffusion coefficients, resulting in relatively high-resolution sedimentation coefficient distributions c(s) or molar mass distributions c(M). It can be applied to interference optical data that exhibit systematic noise components, and it does not require solution or solvent plateaus to be established. More details on the size-distribution can be obtained than from van Holde-Weischet analysis. The sensitivity to the values of the regularization parameter and to the shape parameters is explored with the help of simulated sedimentation data of discrete and continuous model size distributions, and by applications to experimental data of continuous and discrete protein mixtures.
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            Estimation of protein secondary structure from circular dichroism spectra: comparison of CONTIN, SELCON, and CDSSTR methods with an expanded reference set.

             N Sreerama,  R D Woody (2000)
            We have expanded the reference set of proteins used in SELCON3 by including 11 additional proteins (selected from the reference sets of Yang and co-workers and Keiderling and co-workers). Depending on the wavelength range and whether or not denatured proteins are included in the reference set, five reference sets were constructed with the number of reference proteins varying from 29 to 48. The performance of three popular methods for estimating protein secondary structure fractions from CD spectra (implemented in software packages CONTIN, SELCON3, and CDSSTR) and a variant of CONTIN, CONTIN/LL, that incorporates the variable selection method in the locally linearized model in CONTIN, were examined using the five reference sets described here, and a 22-protein reference set. Secondary structure assignments from DSSP were used in the analysis. The performances of all three methods were comparable, in spite of the differences in the algorithms used in the three software packages. While CDSSTR performed the best with a smaller reference set and larger wavelength range, and CONTIN/LL performed the best with a larger reference set and smaller wavelength range, the performances for individual secondary structures were mixed. Analyzing protein CD spectra using all three methods should improve the reliability of predicted secondary structural fractions. The three programs are provided in CDPro software package and have been modified for easier use with the different reference sets described in this paper. CDPro software is available at the website: http://lamar.colostate.edu/ approximately sreeram/CDPro. Copyright 2000 Academic Press.
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              Global rigid body modeling of macromolecular complexes against small-angle scattering data.

              New methods to automatically build models of macromolecular complexes from high-resolution structures or homology models of their subunits or domains against x-ray or neutron small-angle scattering data are presented. Depending on the complexity of the object, different approaches are employed for the global search of the optimum configuration of subunits fitting the experimental data. An exhaustive grid search is used for hetero- and homodimeric particles and for symmetric oligomers formed by identical subunits. For the assemblies or multidomain proteins containing more then one subunit/domain per asymmetric unit, heuristic algorithms based on simulated annealing are used. Fast computational algorithms based on spherical harmonics representation of scattering amplitudes are employed. The methods allow one to construct interconnected models without steric clashes, to account for the particle symmetry and to incorporate information from other methods, on distances between specific residues or nucleotides. For multidomain proteins, addition of missing linkers between the domains is possible. Simultaneous fitting of multiple scattering patterns from subcomplexes or deletion mutants is incorporated. The efficiency of the methods is illustrated by their application to complexes of different types in several simulated and practical examples. Limitations and possible ambiguity of rigid body modeling are discussed and simplified docking criteria are provided to rank multiple models. The methods described are implemented in publicly available computer programs running on major hardware platforms.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2012
                19 September 2012
                : 7
                : 9
                Affiliations
                [1 ]Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria, Australia
                [2 ]Patrys Ltd, Melbourne, Victoria, Australia
                [3 ]Patrys GmbH, Würzburg, Germany
                Duke University Medical Center, United States of America
                Author notes

                Competing Interests: This work is partially supported by Patrys Ltd, a company currently conducting clinical trials of PAT-SM6 as a potential anti-cancer treatment.

                Conceived and designed the experiments: ZR TDM DMH LLI BEP CH FH GJH YFM. Performed the experiments: ZR TDM CH YFM. Analyzed the data: ZR TDM GJH YFM. Contributed reagents/materials/analysis tools: LLI BEP CH FH. Wrote the paper: ZR TDM GJH YFM.

                [¤]

                Current address: Horizon Science Pty Ltd, Braeside, Victoria, Australia

                Article
                PONE-D-12-18562
                10.1371/journal.pone.0044927
                3446985
                23028685

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                Page count
                Pages: 11
                Funding
                This research was supported by the Australian Research Council (LP100100392) with additional support provided by Patrys Ltd. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology
                Biochemistry
                Biophysics
                Immunology
                Immunity
                Immunotherapy
                Molecular Cell Biology
                Signal Transduction
                Signaling Cascades
                Apoptotic Signaling Cascade
                Signaling in Cellular Processes
                Apoptotic Signaling
                Medicine
                Clinical Immunology
                Immunity
                Immunotherapy
                Oncology
                Cancer Treatment
                Immunotherapy

                Uncategorized

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