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Biomedical subjects

Peter Schuck

Publications and source records attributed to Peter Schuck.

At least 37 records · Page 2Linked to original sources
PubMed2005

Targeted lysis of HIV-infected cells by natural killer cells armed and triggered by a recombinant immunoglobulin fusion protein: implications for immunotherapy.

Natural killer (NK) cells play an important role in both innate and adaptive antiviral immune responses. The adaptive response typically requires that virus-specific antibodies decorate infected cells which then direct NK cell lysis through a CD16 mediated process termed antibody-dependent cellular cytotoxicity (ADCC). In this report, we employ a highly polymerized chimeric IgG1/IgA immunoglobulin (Ig) fusion protein that, by virtue of its capacity to extensively crosslink CD16, activates NK cells while directing the lysis of infected target cells. We employ HIV as a model system, and demonstrate that freshly isolated NK cells preloaded with an HIV gp120-specific chimeric IgG1/IgA fusion protein efficiently lyse HIV-infected target cells at picomolar concentrations. NK cells pre-armed in this manner retain the capacity to kill targets over an extended period of time. This strategy may have application to other disease states including various viral infections and cancers.

Antibody-Dependent Cell Cytotoxicity↗
PubMed2005

Cooperative behavior of Escherichia coli cell-division protein FtsZ assembly involves the preferential cyclization of long single-stranded fibrils.

A mechanism of noncooperative (isodesmic) assembly coupled with preferential cyclization of long polymers is proposed to explain the previously posed question of how a single-stranded filament of the bacterial cell-division protein FtsZ can assemble in an apparently cooperative manner. This proposal is based on results of GTP-mediated assembly of FtsZ from Escherichia coli that was studied under physiologically relevant steady-state solution conditions by a combination of methods including measurement of sedimentation velocity, atomic force and electron microscopy, and precipitation assays. Sedimentation-velocity experiments carried out at multiple protein concentrations reveal an essentially bimodal distribution of slowly sedimenting species and a relatively narrow distribution of rapidly sedimenting species that appears only above an apparent "critical concentration" of protein. In a precipitation assay, the amount of protein that pellets, which correlates with the fraction of rapidly sedimenting species observed in sedimentation-velocity experiments, increases linearly with the total concentration of protein in excess of the critical concentration. Sedimentation coefficients of the rapidly sedimenting fraction are qualitatively consistent with the presence of single-stranded cyclic oligomers with a size range of approximately 50-150 protomers, similar to polymeric single-stranded rings observed in atomic force and electron micrographs. The proposed model is in accord with the results obtained from our experimental observations.

Centrifugation↗
PubMed2005

Exposure of acidic residues as a danger signal for recognition of fibrinogen and other macromolecules by integrin alphaXbeta2.

The structural integrity of tissue proteins is damaged in processes ranging from remodeling of the extracellular matrix to destruction by microbial pathogens. Leukocytes play a prominent role in tissue surveillance and repair. However, it remains enigmatic what features of structurally decayed proteins prompt recognition by leukocyte cell-surface receptors. Here, we report that adhesion of human neutrophil granulocytes to fibrinogen is greatly increased by plasmin digestion in a mode where alphaXbeta2 dominates the integrin-dependent binding. The bacterial protease subtilisin also enhances binding by alphaXbeta2. The alphaX ligand binding domain has an unusually high affinity for carboxyl groups, with KD at approximately 100 microM. Our findings implicate enhanced accessibility of negatively charged residues in structurally decayed proteins as a pattern recognition motif for alphaXbeta2 integrin. Comparisons among integrins show relevance of these findings to the large number of ligands recognized by alphaMbeta2 and alphaXbeta2 but not alphaLbeta2. The observations suggest that the pericellular proteolysis at the leading edge of neutrophils not only facilitates passage through the extracellular matrix but also manufactures binding sites for alphaXbeta2.

Binding Sites↗
PubMed2005

Characterization of DC-SIGN/R interaction with human immunodeficiency virus type 1 gp120 and ICAM molecules favors the receptor's role as an antigen-capturing rather than an adhesion receptor.

The dendritic cell (DC)-specific intercellular adhesion molecule 3 (ICAM-3)-grabbing nonintegrin binding receptor (DC-SIGN) was shown to bind human immunodeficiency virus type 1 (HIV-1) viral envelope protein gp120 and proposed to function as a Trojan horse to enhance trans-virus infection to host T cells. To better understand the mechanism by which DC-SIGN and DC-SIGNR selectively bind HIV-1 gp120, we constructed a series of deletion mutations in the repeat regions of both receptors. Different truncated receptors exist in different oligomeric forms. The carbohydrate binding domain without any repeats was monomeric, whereas the full extracellular receptors existed as tetramers. All reconstituted receptors retained their ability to bind gp120. The dissociation constant, however, differed drastically from micromolar values for the monomeric receptors to nanomolar values for the tetrameric receptors, suggesting that the repeat region of these receptors contributes to the avidity of gp120 binding. Such oligomerization may provide a mechanism for the receptor to selectively recognize pathogens containing multiple high-mannose-concentration carbohydrates. In contrast, the receptors bound to ICAMs with submicromolar affinities that are similar to those of two nonspecific cell surface glycoproteins, FcgammaRIIb and FcgammaRIII, and the oligomerization of DC-SIGNR resulted in no increase in binding affinity to ICAM-3. These findings suggest that DC-SIGN may not discriminate other cell surface glycoproteins from ICAM-3 binding. The pH dependence in DC-SIGN binding to gp120 showed that the receptor retained high-affinity gp120 binding at neutral pH but lost gp120 binding at pH 5, suggesting a release mechanism of HIV in the acidic endosomal compartment by DC-SIGN. Our work contradicts the function of DC-SIGN as a Trojan horse to facilitate HIV-1 infection; rather, it supports the function of DC-SIGN/R (a designation referring to both DC-SIGN and DC-SIGNR) as an antigen-capturing receptor.

Animals↗
PubMed2004

Studying multiprotein complexes by multisignal sedimentation velocity analytical ultracentrifugation.

Protein interactions can promote the reversible assembly of multiprotein complexes, which have been identified as critical elements in many regulatory processes in cells. The biophysical characterization of assembly products, their number and stoichiometry, and the dynamics of their interactions in solution can be very difficult. A classical first-principle approach for the study of purified proteins and their interactions is sedimentation velocity analytical ultracentrifugation. This approach allows one to distinguish different protein complexes based on their migration in the centrifugal field without isolating reversibly formed complexes from the individual components. An important existing limitation for systems with multiple components and assembly products is the identification of the species associated with the observed sedimentation rates. We developed a computational approach for integrating multiple optical signals into the sedimentation coefficient distribution analysis of components, which combines the size-dependent hydrodynamic separation with discrimination of the extinction properties of the sedimenting species. This approach allows one to deduce the stoichiometry and to assign the identity of the assembly products without prior assumptions of the number of species and the nature of their interaction. Although chromophoric labels may be used to enhance the spectral resolution, we demonstrate the ability to work label-free for three-component protein mixtures. We observed that the spectral discrimination can synergistically enhance the hydrodynamic resolution. This method can take advantage of differences in the absorbance spectra of interacting solution components, for example, for the study of protein-protein, protein-nucleic acid or protein-small molecule interactions, and can determine the size, hydrodynamic shape, and stoichiometry of multiple complexes in solution.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Crystal structure of the C-terminal peptidoglycan-binding domain of human peptidoglycan recognition protein Ialpha.

Peptidoglycan recognition proteins (PGRPs) are pattern recognition receptors of the innate immune system that bind, and in some cases hydrolyze, peptidoglycans (PGNs) on bacterial cell walls. These molecules, which are highly conserved from insects to mammals, participate in host defense against both Gram-positive and Gram-negative bacteria. We report the crystal structure of the C-terminal PGN-binding domain of human PGRP-Ialpha in two oligomeric states, monomer and dimer, to resolutions of 2.80 and 1.65 A, respectively. In contrast to PGRPs with PGN-lytic amidase activity, no zinc ion is present in the PGN-binding site of human PGRP-Ialpha. The structure reveals that PGRPs exhibit extensive topological variability in a large hydrophobic groove, located opposite the PGN-binding site, which may recognize host effector proteins or microbial ligands other than PGN. We also show that full-length PGRP-Ialpha comprises two tandem PGN-binding domains. These domains differ at most potential PGN-contacting positions, implying different fine specificities. Dimerization of PGRP-Ialpha, which occurs through three-dimensional domain swapping, is mediated by specific binding of sodium ions to a flexible hinge loop, stabilizing the conformation found in the dimer. We further demonstrate sodium-dependent dimerization of PGRP-Ialpha in solution, suggesting a possible mechanism for modulating PGRP activity through the formation of multivalent adducts.

Amino Acid Sequence↗
PubMed2004

Binding specificity of multiprotein signaling complexes is determined by both cooperative interactions and affinity preferences.

The generation of multiprotein complexes at receptors and adapter proteins is crucial for the activation of intracellular signaling pathways. In this study, we used multiple biochemical and biophysical methods to examine the binding properties of several SH2 and SH3 domain-containing signaling proteins as they interact with the adapter protein linker for activation of T-cells (LAT) to form multiprotein complexes. We observed that the binding specificity of these proteins for various LAT tyrosines appears to be constrained both by the affinity of binding and by cooperative protein-protein interactions. These studies provide quantitative information on how different binding parameters can determine in vivo binding site specificity observed for multiprotein signaling complexes.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Sedimentation equilibrium analysis of protein interactions with global implicit mass conservation constraints and systematic noise decomposition.

Sedimentation equilibrium is a powerful tool for the characterization of protein self-association and heterogeneous protein interactions. Frequently, it is applied in a configuration with relatively long solution columns and with equilibrium profiles being acquired sequentially at several rotor speeds. The present study proposes computational tools, implemented in the software SEDPHAT, for the global analysis of equilibrium data at multiple rotor speeds with multiple concentrations and multiple optical detection methods. The detailed global modeling of such equilibrium data can be a nontrivial computational problem. It was shown previously that mass conservation constraints can significantly improve and extend the analysis of heterogeneous protein interactions. Here, a method for using conservation of mass constraints for the macromolecular redistribution is proposed in which the effective loading concentrations are calculated from the sedimentation equilibrium profiles. The approach is similar to that described by Roark (Biophys. Chem. 5 (1976) 185-196), but its utility is extended by determining the bottom position of the solution columns from the macromolecular redistribution. For analyzing heterogeneous associations at multiple protein concentrations, additional constraints that relate the effective loading concentrations of the different components or their molar ratio in the global analysis are introduced. Equilibrium profiles at multiple rotor speeds also permit the algebraic determination of radial-dependent baseline profiles, which can govern interference optical ultracentrifugation data, but usually also occur, to a smaller extent, in absorbance optical data. Finally, the global analysis of equilibrium profiles at multiple rotor speeds with implicit mass conservation and computation of the bottom of the solution column provides an unbiased scale for determining molar mass distributions of noninteracting species. The properties of these tools are studied with theoretical and experimental data sets.

Macromolecular Substances↗
PubMed2004

A model for sedimentation in inhomogeneous media. I. Dynamic density gradients from sedimenting co-solutes.

Macromolecular sedimentation in inhomogeneous media is of great practical importance. Dynamic density gradients have a long tradition in analytical ultracentrifugation, and are frequently used in preparative ultracentrifugation. In this paper, a new theoretical model for sedimentation in inhomogeneous media is presented, based on finite element solutions of the Lamm equation with spatial and temporal variation of the local solvent density and viscosity. It is applied to macromolecular sedimentation in the presence of a dynamic density gradient formed by the sedimentation of a co-solute at high concentration. It is implemented in the software SEDFIT for the analysis of experimental macromolecular concentration distributions. The model agrees well with the measured sedimentation profiles of a protein in a dynamic cesium chloride gradient, and may provide a measure for the effects of hydration or preferential solvation parameters. General features of protein sedimentation in dynamic density gradients are described.

Attention↗
PubMed2004

A model for sedimentation in inhomogeneous media. II. Compressibility of aqueous and organic solvents.

The effects of solvent compressibility on the sedimentation behavior of macromolecules as observed in analytical ultracentrifugation are examined. Expressions for the density and pressure distributions in the solution column are derived and combined with the finite element solution of the Lamm equation in inhomogeneous media to predict the macromolecular concentration distributions under different conditions. Independently, analytical expressions are derived for the sedimentation of non-diffusing particles in the limit of low compressibility. Both models are quantitatively consistent and predict solvent compressibility to result in a reduction of the sedimentation rate along the solution column and a continuous accumulation of solutes in the plateau region. For both organic and aqueous solvents, the calculated deviations from the sedimentation in incompressible media can be very large and substantially above the measurement error. Assuming conventional configurations used for sedimentation velocity experiments in analytical ultracentrifugation, neglect of the compressibility of water leads to systematic errors underestimating sedimentation coefficients by approximately 1% at a rotor speeds of 45000 rpm, but increasing to 2-5% with increasing rotor speeds and decreasing macromolecular size. The proposed finite element solution of the Lamm equation can be used to take solvent compressibility quantitatively into account in direct boundary models for discrete species, sedimentation coefficient distributions or molar mass distributions. Using the analytical expressions for the sedimentation of non-diffusing particles, the ls-g*(s) distribution of apparent sedimentation coefficients is extended to the analysis of sedimentation in compressible solvents. The consideration of solvent compressibility is highly relevant not only when using organic solvents, but also in aqueous solvents when precise sedimentation coefficients are needed, for example, for hydrodynamic modeling.

Centrifugation, Density Gradient↗
PubMed2004

SDR grafting of a murine antibody using multiple human germline templates to minimize its immunogenicity.

The humanization of mAbs by complementarity-determining region (CDR)-grafting has become a standard procedure to improve the clinical utility of xenogeneic Abs by reducing human anti-murine Ab (HAMA) responses elicited in patients. However, CDR-grafted humanized Abs may still evoke anti-V region responses when administered in patients. To minimize anti-V region responses, the Ab may be humanized by grafting onto the human templates only the specificity-determining residues (SDRs), the residues that are essential for the surface complementarity of the Ab and its ligand. Typically, humanization of an Ab, whether by CDR or SDR grafting, involves the use of a single human template for the entire VL or VH domain of an Ab. We hypothesized, however, that the homology between the human template sequences and mAb to be humanized may be maximized by using templates from multiple human germline sequences corresponding to the different segments of the variable domain. This could be more advantageous in reducing the potential immunogenicity of the humanized Ab. This report describes the SDR grafting of the murine anti-carcinoembryonic antigen (CEA) mAb COL-1 using three different human germline V-kappa sequences as templates for the VL CDRs and another human template for the VL frameworks. In competition RIAs, the SDR-grafted COL-1 (HuCOL-1SDR) completely inhibited the binding of radiolabeled murine COL-1 (mCOL-1) to CEA, and showed that its binding affinity is comparable to that of the CDR-grafted Ab (HuCOL-1). The HuCOL-1SDR showed similar binding reactivity to the CEA expressed on the surface of a tumor cell line as the HuCOL-1. More importantly, compared to HuCOL-1 and the "abbreviated" CDR-grafted Ab, HuCOL-1SDR showed lower reactivity to patients' sera carrying anti-V region Abs to mCOL-1. HuCOL-1SDR, which shows a lower sera reactivity than that of the parental Abs while retaining its Ag-binding property, is a potentially useful clinical reagent. To the best of our knowledge, this is the first time a VL or VH domain of an Ab has been humanized by grafting the SDRs onto a human template comprised of several Ab sequences. We have shown that humanization of an Ab can be optimized using multiple human templates for a single variable domain of an Ab. This approach maximizes the homology between the target Ab and the human templates in both the frameworks and the CDRs by choosing as the template the human sequence that displays the highest local sequence identity to the frameworks and to each of the CDRs of the target Ab.

Amino Acids↗
PubMed2004

Assessing reproducibility for interval data in health-related quality of life questionnaires: which coefficient should be used?

In contrast to other reliability estimates, test-retest reliability (or reproducibility) captures not only the measurement error of an assessment instrument, but also the stability of the construct measured. Consequently, one would expect any departure from identity (Y = X) of measurement pairs (X first, and Y second measurement) to be treated as 'error' by the respective reproducibility statistic, even if 'true' changes happened, e.g. worsening of a disease due to its natural course. The Pearson correlation, still often advocated for continuous measures in test-retest reliability studies, however captures the degree of linearity (Y = bX + a): perfect relationship can be computed, even if the measurement pairs differ not only by a additive constant 'a', but also because of a multiplication of the X-values with the slope 'b'. Therefore, intraclass correlation coefficients (ICCs) have been proposed as alternative statistics for reproducibility. However, only ICCs with absolute agreement definition of concordance capture the degree of identity. ICCs with a consistency definition of concordance measure the degree of additivity (Y = X + a). ICCs are calculated from repeated measures analyses of variance (ANOVAs), and a common population variance must be is assumed for the different measurements. Given this assumption, an ICC computed from a one-way ANOVA seems to be the best choice for this purpose. Otherwise, Lin's concordance correlation coefficient is recommended as identity measure.

Analysis of Variance↗
PubMed2004

Case histories illustrating the utility of sertindole in clinical practice.

Many conventional and atypical antipsychotic agents are available for the treatment of schizophrenia. Matching individual patients to the medication that suits them best is often a matter of trial and error. Controlled clinical trials and extensive post-marketing research have shown that the atypical antipsychotic agent sertindole is a broadly efficacious and safe choice for the treatment of schizophrenia. Individual case reports emerging from the ongoing sertindole post-marketing clinical programme illustrate how, under normal conditions of clinical practice, a switch to sertindole can benefit patients who have, for various reasons, previously not been satisfactorily managed on conventional or other atypical antipsychotic agents. Such case reports have shown that a switch to sertindole has the potential to provide greater relief from positive, negative and affective symptoms than has been achieved with conventional and certain other atypical agents. Cognitive dysfunction evident during therapy with conventional antipsychotic agents has also been seen to improve in individuals switched to sertindole. These improvements have occurred without evidence of extrapyramidal symptoms or other poorly tolerated adverse events. After switching to sertindole, the five patients described in this report complied well with therapy, were generally able to perform daily activities more efficiently, and also had improved personal relationships and vocational potential, all of which contributed to improvements in their overall quality of life.

Adult↗
PubMed2003

In vitro affinity maturation of a specificity-determining region-grafted humanized anticarcinoma antibody: isolation and characterization of minimally immunogenic high-affinity variants.

PURPOSE: HuCC49V10 (V10), a humanized anticarcinoma monoclonal antibody (Ab) CC49, was generated by grafting only the specificity-determining regions (SDRs) of CC49 onto the variable light and variable heavy frameworks of the human Abs LEN and 21/28'CL, respectively. SDRs are those residues of the complementarity-determining regions that are most critical for antigen (Ag) binding. Compared with HuCC49, which was developed by conventional complementarity-determining region grafting, V10 has lower reactivity to the sera from patients who were previously given murine CC49 in clinical trials, although its Ag-binding affinity is 2-3-fold lower than that of HuCC49. To generate variants of V10 with higher Ag-binding affinity and lower sera reactivity, in vitro affinity maturation of V10 was carried out using phage display technique. EXPERIMENTAL DESIGN: A limited library of Fabs was generated by replacing some of the SDRs with all possible residues located at the corresponding positions in human Abs. The library was enriched, by several rounds of panning, in Fabs that have high affinity for the TAG-72 Ag. The clones encoding the best binders were expressed in insect cells as whole Abs that were purified and characterized. RESULTS: Competition radioimmunoassay and surface plasmon resonance measurements showed that two of the isolates, V14 and V15, have higher binding affinity than that of V10. In addition, the surface plasmon resonance analysis showed that the variants V14 and V15, compared with the parental V10, have lower reactivity to the anti-V region Abs using sera from patients who received murine CC49. CONCLUSIONS: The two isolates, V14 and V15, which show higher Ag-binding reactivity and lower sera reactivity than the parental V10 Ab, are potentially more useful clinical reagents. These results demonstrate that phage display can be used to isolate variants of an Ab that are potentially less immunogenic in patients than the parental Ab from which they are derived.

Adenocarcinoma↗
PubMed2003

Variable MHC class I engagement by Ly49 natural killer cell receptors demonstrated by the crystal structure of Ly49C bound to H-2K(b).

The Ly49 family of natural killer (NK) receptors regulates NK cell function by sensing major histocompatibility complex (MHC) class I. Ly49 receptors show complex patterns of MHC class I cross-reactivity and, in certain cases, peptide selectivity. To investigate whether specificity differences result from topological differences in MHC class I engagement, we determined the structure of the peptide-selective receptor Ly49C in complex with H-2K(b). The Ly49C homodimer binds two MHC class I molecules in symmetrical way, a mode distinct from that of Ly49A, which binds MHC class I asymmetrically. Ly49C does not directly contact the MHC-bound peptide. In addition, MHC crosslinking by Ly49C was demonstrated in solution. We propose a dynamic model for Ly49-MHC class I interactions involving conformational changes in the receptor, whereby variations in Ly49 dimerization mediate different MHC-binding modes.

Animals↗
PubMed2003

On the analysis of protein self-association by sedimentation velocity analytical ultracentrifugation.

Analytical ultracentrifugation is one of the classical techniques for the study of protein interactions and protein self-association. Recent instrumental and computational developments have significantly enhanced this methodology. In this paper, new tools for the analysis of protein self-association by sedimentation velocity are developed, their statistical properties are examined, and considerations for optimal experimental design are discussed. A traditional strategy is the analysis of the isotherm of weight-average sedimentation coefficients s(w) as a function of protein concentration. From theoretical considerations, it is shown that integration of any differential sedimentation coefficient distribution c(s), ls-g(*)(s), or g(s(*)) can give a thermodynamically well-defined isotherm, as long as it provides a good model for the sedimentation profiles. To test this condition for the g(s(*)) distribution, a back-transform into the original data space is proposed. Deconvoluting diffusion in the sedimentation coefficient distribution c(s) can be advantageous to identify species that do not participate in the association. Because of the large number of scans that can be analyzed in the c(s) approach, its s(w) values are very precise and allow extension of the isotherm to very low concentrations. For all differential sedimentation coefficients, corrections are derived for the slowing of the sedimentation boundaries caused by radial dilution. As an alternative to the interpretation of the isotherm of the weight-average s value, direct global modeling of several sedimentation experiments with Lamm equation solutions was studied. For this purpose, a new software SEDPHAT is introduced, allowing the global analysis of several sedimentation velocity and equilibrium experiments. In this approach, information from the shape of the sedimentation profiles is exploited, which permits the identification of the association scheme and requires fewer experiments to precisely characterize the association. Further, under suitable conditions, fractions of incompetent material that are not part of the reversible equilibrium can be detected.

Algorithms↗
PubMed2003

Alanine-scanning mutations in domain 4 of anthrax toxin protective antigen reveal residues important for binding to the cellular receptor and to a neutralizing monoclonal antibody.

A panel of variants with alanine substitutions in the small loop of anthrax toxin protective antigen domain 4 was created to determine individual amino acid residues critical for interactions with the cellular receptor and with a neutralizing monoclonal antibody, 14B7. Substituted protective antigen proteins were analyzed by cellular cytotoxicity assays, and their interactions with antibody were measured by plasmon surface resonance and analytical ultracentrifugation. Residue Asp683 was the most critical for cell binding and toxicity, causing an approximately 1000-fold reduction in toxicity, but was not a large factor for interactions with 14B7. Substitutions in residues Tyr681, Asn682, and Pro686 also reduced toxicity significantly, by 10-100-fold. Of these, only Asn682 and Pro686 were also critical for interactions with 14B7. However, residues Lys684, Leu685, Leu687, and Tyr688 were critical for 14B7 binding without greatly affecting toxicity. The K684A and L685A variants exhibited wild type levels of toxicity in cell culture assays; the L687A and Y688A variants were reduced only 1.5- and 5-fold, respectively.

Alanine↗