Project description:The SuperPose web server rapidly and robustly calculates both pairwise and multiple protein structure superpositions using a modified quaternion eigenvalue approach. SuperPose generates sequence alignments, structure alignments, PDB (Protein Data Bank) coordinates and RMSD statistics, as well as difference distance plots and images (both static and interactive) of the superimposed molecules. SuperPose employs a simple interface that requires only PDB files or accession numbers as input. All other superposition decisions are made by the program. SuperPose is uniquely able to superimpose structures that differ substantially in sequence, size or shape. It is also capable of handling a much larger range of superposition queries and situations than many standalone programs and yields results that are intuitively more in agreement with known biological or structural data. The SuperPose web server is freely accessible at http://wishart.biology.ualberta.ca/SuperPose/.
Project description:NKT cells recognize lipid Ags presented by CD1d molecules and play an important role in the regulation of innate and adaptive immune responses. In this study, we report the identification of a membrane-associated protein, Ig-like transcript 4 (ILT4), as a novel human CD1d receptor that inhibits CD1d-mediated immune responses. We found that native CD1d tetramer generated by mammalian cells was able to specifically bind human monocytes in the peripheral blood, and this binding was at least partly mediated by monocyte-expressed ILT4. The interaction between ILT4 and CD1d involves the two N-terminal domains of ILT4 and the Ag-binding groove of CD1d (alpha1/alpha2 domain). This interaction has been identified on the cell surface as well as in the endosomal and lysosomal compartments. Functional analysis showed that ILT4 could block the loading of lipid Ags such as alpha-GalCer, and consequently inhibited NKT recognition. The interaction between ILT4 and CD1d may provide new insights into the regulation of NKT-mediated immunity.
Project description:BackgroundProtein A, protein G and protein L are three well-defined immunoglobulin (Ig)-binding proteins (IBPs), which show affinity for specific sites on Ig of mammalian hosts. Although the precise functions of these molecules are not fully understood, it is thought that they play an important role in pathogenicity of bacteria. The single domains of protein A, protein G and protein L were all demonstrated to have function to bind to Ig. Whether combinations of Ig-binding domains of various IBPs could exhibit useful novel binding is interesting.ResultsWe used a combinatorial phage library which displayed randomly-rearranged various-peptide-linked molecules of D and A domains of protein A, designated PA(D) and PA(A) respectively, B2 domain of protein G (PG) and B3 domain of protein L (PL) for affinity selection with human IgG (hIgG), human IgM (hIgM), human IgA (hIgA) and recombinant hIgG1-Fc as bait respectively. Two kinds of novel combinatorial molecules with characteristic structure of PA(A)-PG and PA(A)-PL were obtained in hIgG (hIgG1-Fc) and hIgM (hIgA) post-selection populations respectively. In addition, the linking peptides among all PA(A)-PG and PA(A)-PL structures was strongly selected, and showed interestingly divergent and convergent distribution. The phage binding assays and competitive inhibition experiments demonstrated that PA(A)-PG and PA(A)-PL combinations possess comparable binding advantages with hIgG/hIgG1-Fc and hIgM/hIgA respectively.ConclusionIn this work, a combinatorial phage library displaying Ig-binding domains of protein A, protein G, or protein L joined by various random linking peptides was used to conducted evolutional selection in vitro with four kinds of Ig molecules. Two kinds of novel combinations of Ig-binding domains, PA(A)-PG and PA(A)-PL, were obtained, and demonstrate the novel Ig binding properties.
Project description:Chimeric antigen receptor (CAR)-expressing T cells targeting B-cell maturation antigen (BCMA) have activity against multiple myeloma, but improvements in anti-BCMA CARs are needed. We demonstrated recipient anti-CAR T-cell responses against a murine single-chain variable fragment (scFv) used clinically in anti-BCMA CARs. To bypass potential anti-CAR immunogenicity and to reduce CAR binding domain size, here we designed CARs with antigen-recognition domains consisting of only a fully human heavy-chain variable domain without a light-chain domain. A CAR designated FHVH33-CD8BBZ contains a fully human heavy-chain variable domain (FHVH) plus 4-1BB and CD3ζ domains. T cells expressing FHVH33-CD8BBZ exhibit similar cytokine release, degranulation, and mouse tumor eradication as a CAR that is identical except for substitution of a scFv for FHVH33. Inclusion of 4-1BB is critical for reducing activation-induced cell death and promoting survival of T cells expressing FHVH33-containing CARs. Our results indicate that heavy-chain-only anti-BCMA CARs are suitable for evaluation in a clinical trial.
Project description:Chimeric antigen receptors (CARs) are artificial fusion proteins that incorporate antigen-recognition domains and T cell signaling domains. CD30 is a cell surface protein expressed on Hodgkin's lymphoma, some T cell lymphomas, and some B cell lymphomas. CD30 has a restricted expression pattern in normal cells, so CD30 has good potential as a clinical target for CAR T cells. We compared three different anti-CD30 CAR designs incorporating a single-chain variable fragment derived from the 5F11 fully human monoclonal antibody. 5F11-28Z has hinge, transmembrane, and costimulatory domains from CD28 and a CD3ζ T cell activation domain. 5F11-CD828Z has hinge and transmembrane domains from CD8α, a CD28 costimulatory domain, and a CD3ζ T cell activation domain. 5F11-CD8BBZ is identical to 5F11-CD828Z, except for the replacement of the CD28 moiety with a 4-1BB moiety. We found that T cells expressing 5F11-CD8BBZ had lower levels of CD30-specific degranulation and cytokine release compared with CD28-containing CARs. When compared to the CD28-containing CARs, T cells expressing 5F11-CD8BBZ had higher levels of nonspecific functional activity, including degranulation, cytokine release, and proliferation, when stimulated with CD30-negative target cells. We established tumors in nod-scid common gamma-chain deficient (NSG) mice and treated the tumors with T cells expressing different CARs. T cells expressing 5F11-28Z were most effective at eradicating tumors. T cells expressing 5F11-CD828Z had intermediate effectiveness, and T cells expressing 5F11-CD8BBZ were least effective. CD30+ T cells are lost from cultures of T cells containing 5F11-28Z-expressing T cells. This indicated the killing of CD30+ T cells by the 5F11-28Z-expressing T cells. Despite this, the number of T cells in the cultures consistently accumulated to numbers needed for use in a clinical trial. Based on all in vitro and murine experiments comparing the different CARs, we selected 5F11-28Z for further development, and we have initiated a clinical trial testing 5F11-28Z T cells.
Project description:Targeting protein stability with small molecules has emerged as an effective tool to control protein abundance in a fast, scalable and reversible manner. The technique involves tagging a protein of interest (POI) with a destabilizing domain (DD) specifically controlled by a small molecule. The successful construction of such fusion proteins may, however, be limited by functional interference of the DD epitope with electrostatic interactions required for full biological function of proteins. Another drawback of this approach is the remaining endogenous protein. Here, we combined the Cre-LoxP system with an advanced DD and generated a protein regulation system in which the loss of an endogenous protein, in our case the tumor suppressor PTEN, can be coupled directly with a conditionally fine-tunable DD-PTEN. This new system will consolidate and extend the use of DD-technology to control protein function precisely in living cells and animal models.
Project description:Variable lymphocyte receptors (VLRs) are unconventional adaptive immune receptors relatively recently discovered in the phylogenetically ancient jawless vertebrates, lamprey and hagfish. VLRs bind antigens using a leucine-rich repeat fold and are the only known adaptive immune receptors that do not utilize an immunoglobulin fold for antigen recognition. While immunoglobulin antibodies have been studied extensively, there are comparatively few studies on antigen recognition by VLRs, particularly for protein antigens. Here we report isolation, functional and structural characterization of three VLRs that bind the protein toll-like receptor 5 (TLR5) from zebrafish. Two of the VLRs block binding of TLR5 to its cognate ligand flagellin in functional assays using reporter cells. Co-crystal structures revealed that these VLRs bind to two different epitopes on TLR5, both of which include regions involved in flagellin binding. Our work here demonstrates that the lamprey adaptive immune system can be used to generate high-affinity VLR clones that recognize different epitopes and differentially impact natural ligand binding to a protein antigen.
Project description:The emergence of immune escape is a significant roadblock to developing effective chimeric antigen receptor (CAR) T cell therapies against hematological malignancies, including acute myeloid leukemia (AML). Here, we demonstrate feasibility of targeting two antigens simultaneously by combining a GRP78-specific peptide antigen recognition domain with a CD123-specific scFv to generate a peptide-scFv bispecific antigen recognition domain (78.123). To achieve this, we test linkers with varying length and flexibility and perform immunophenotypic and functional characterization. We demonstrate that bispecific CAR T cells successfully recognize and kill tumor cells that express GRP78, CD123, or both antigens and have improved antitumor activity compared to their monospecific counterparts when both antigens are expressed. Protein structure prediction suggests that linker length and compactness influence the functionality of the generated bispecific CARs. Thus, we present a bispecific CAR design strategy to prevent immune escape in AML that can be extended to other peptide-scFv combinations.
Project description:Decision-making animals can use slow-but-accurate strategies, such as making multiple comparisons, or opt for simpler, faster strategies to find a 'good enough' option. Social animals make collective decisions about many group behaviours including foraging and migration. The key to the collective choice lies with individual behaviour. We present a case study of a collective decision-making process (house-hunting ants, Temnothorax albipennis), in which a previously proposed decision strategy involved both quality-dependent hesitancy and direct comparisons of nests by scouts. An alternative possible decision strategy is that scouting ants use a very simple quality-dependent threshold rule to decide whether to recruit nest-mates to a new site or search for alternatives. We use analytical and simulation modelling to demonstrate that this simple rule is sufficient to explain empirical patterns from three studies of collective decision-making in ants, and can account parsimoniously for apparent comparison by individuals and apparent hesitancy (recruitment latency) effects, when available nests differ strongly in quality. This highlights the need to carefully design experiments to detect individual comparison. We present empirical data strongly suggesting that best-of-n comparison is not used by individual ants, although individual sequential comparisons are not ruled out. However, by using a simple threshold rule, decision-making groups are able to effectively compare options, without relying on any form of direct comparison of alternatives by individuals. This parsimonious mechanism could promote collective rationality in group decision-making.
Project description:Molecular elasticity is associated with a select number of polypeptides and proteins, such as titin, Lustrin A, silk fibroin, and spider silk dragline protein. In the case of titin, the globular (Ig) and non-globular (PEVK) regions act as extensible springs under stretch; however, their unfolding behavior and force extension characteristics are different. Using our time-dependent macroscopic method for simulating AFM-induced titin Ig domain unfolding and refolding, we simulate the extension and relaxation of hypothetical titin chains containing Ig domains and a PEVK region. Two different models are explored: 1) a series-linked WLC expression that treats the PEVK region as a distinct entropic spring, and 2) a summation of N single WLC expressions that simulates the extension and release of a discrete number of parallel titin chains containing constant or variable amounts of PEVK. In addition to these simulations, we also modeled the extension of a hypothetical PEVK domain using a linear Hooke's spring model to account for "enthalpic" contributions to PEVK elasticity. We find that the modified WLC simulations feature chain length compensation, Ig domain unfolding/refolding, and force-extension behavior that more closely approximate AFM, laser tweezer, and immunolocalization experimental data. In addition, our simulations reveal the following: 1) PEVK extension overlaps with the onset of Ig domain unfolding, and 2) variations in PEVK content within a titin chain ensemble lead to elastic diversity within that ensemble.