Biomedical subjects
Publications and source records attributed to B Seed.
Recent studies have identified molecules that interact with integrins and appear to participate in the signaling pathways that regulate integrin adhesiveness. Clues provided by studies of these molecules point to the integration by integrins of signal transduction pathways implicated in cell division and activation.
The CD95 (Fas/APO-1) and tumor necrosis factor (TNF) receptor pathways share many similarities, including a common reliance on proteins containing 'death domains' for elements of the membrane-proximal signal relay. We have created mutant cell lines that are unable to activate NF-kappaB in response to TNF. One of the mutant lines lacks RIP, a 74 kDa Ser/Thr kinase originally identified by its ability to associate with Fas/APO-1 and induce cell death. Reconstitution of the line with RIP restores responsiveness to TNF. The RIP-deficient cell line is susceptible to apoptosis initiated by anti-CD95 antibodies. An analysis of cells reconstituted with mutant forms of RIP reveals similarities between the action of RIP and FADD/MORT-1, a Fas-associated death domain protein.
The avidity of integrin adhesion receptors for extracellular ligands is subject to dynamic regulation by intracellular programs that have yet to be elucidated. We describe here a protein, cytohesin-1, which specifically interacts with the intracellular portion of the integrin beta 2 chain (CD18). The molecule shows homology to the yeast SEC7 gene product and bears a pleckstrin homology (PH) domain. Overexpression of either the full-length cytohesin-1 or the SEC7 domain induces beta 2 integrin-dependent binding of Jurkat cells to ICAM-1, whereas expression of the isolated cytohesin-1 PH domain inhibits T cell receptor-stimulated adhesion. Similar inhibition is not exhibited by PH domains taken from other proteins, showing that the interaction is specific and that individual PH domains are capable of discriminating between alternative targets.
BACKGROUND: The expression of both the env and gag gene products of human immunodeficiency virus type 1 (HIV-1) is known to be limited by cis elements in the viral RNA that impede egress from the nucleus and reduce the efficiency of translation. Identifying these elements has proven difficult, as they appear to be disseminated throughout the viral genome. RESULTS: Here, we report that selective codon usage appears to account for a substantial fraction of the inefficiency of viral protein synthesis, independent of any effect on improved nuclear export. The codon usage effect is not specific to transcripts of HIV-1 origin. Re-engineering the coding sequence of a model protein (Thy-1) with the most prevalent HIV-1 codons significantly impairs Thy-1 expression, whereas altering the coding sequence of the jellyfish green fluorescent protein gene to conform to the favored codons of highly expressed human proteins results in a substantial increase in expression efficiency. CONCLUSIONS: Codon-usage effects are a major impediment to the efficient expression of HIV-1 genes. Although mammalian genes do not show as profound a bias as do Escherichia coli genes, other proteins that are poorly expressed in mammalian cells can benefit from codon re-engineering.
Phagocytosis is a phylogenetically primitive mechanism adapted by specialized cells of the immune system to ingest particulate pathogens. Recent evidence suggests that the program of specific cytoskeletal rearrangements that underlies phagocytosis may share elements with the antigen receptor signaling pathway in lymphocytes. Tyrosine phosphorylation, necessary for both lymphocyte effector function and phagocytosis, is thought to allow cytoskeletal elements to couple to the intracellular domains of antigen and Fc receptor subunits. We show here that the intracellular domains of the receptors are not inherently required for cytoskeletal coupling. Chimeric transmembrane proteins bearing syk but not src family tyrosine kinase domains are capable of autonomously triggering phagocytosis and redistribution of filamentous actin in COS cells. These responses cannot be initiated by a receptor chimera bearing a point mutation in the syk catalytic domain, and the kinase domain alone is sufficient for initiating cytoskeletal coupling.
The XR-1 Chinese hamster ovary cell line is impaired in DNA double-strand break repair (DSBR) and in ability to support V(D)J recombination of transiently introduced substrates. We now show that XR-1 cells support recombination-activating gene 1- and 2-mediated initiation of V(D)J recombination within a chromosomally integrated substrate, but are highly impaired in ability to complete the process by forming coding and recognition sequence joins. On this basis, we isolated a human cDNA sequence, termed XRCC4, whose expression confers normal V(D)J recombination ability and significant restoration of DSBR activity to XR-1, clearly demonstrating that this gene product is involved in both processes. The XRCC4 gene maps to the previously identified locus on human chromosome 5, is deleted in XR-1 cells, and encodes a ubiquitously expressed product unrelated to any described protein.
P-selectin binding to neutrophils requires a specific protein, P-selectin glycoprotein ligand 1 (PSGL-1), as well as sialyl-Lewis X (sLex) glycan determinants. We have found that a short segment near the amino terminus of PSGL-1 that contains a tyrosine sulfation consensus is essential for P-selectin adhesion and that addition of the amino-terminal segment to some but not all mucin-like molecules confers on those molecules the ability to bind P-selectin. PSGL-1 synthesized in the presence of sulfation inhibitors binds P-selectin weakly, and within the amino-terminal 20 residues, mutation of the tyrosines to phenylalanine abolishes binding. Rolling of HL-60 cells on P-selectin-coated coverslips is strongly attenuated by treatment of cells with an inhibitor of sulfation.
Ligation of the extracellular domain of the cell surface receptor Fas/APO-1 (CD95) elicits a characteristic programmed death response in susceptible cells. Using a genetic selection based on protein-protein interaction in yeast, we have identified two gene products that associate with the intracellular domain of Fas: Fas itself, and a novel 74 kDa protein we have named RIP, for receptor interacting protein. RIP also interacts weakly with the p55 tumor necrosis factor receptor (TNFR1) intracellular domain, but not with a mutant version of Fas corresponding to the murine lprcg mutation. RIP contains an N-terminal region with homology to protein kinases and a C-terminal region containing a cytoplasmic motif (death domain) present in the Fas and TNFR1 intracellular domains. Transient overexpression of RIP causes transfected cells to undergo the morphological changes characteristic of apoptosis. Taken together, these properties indicate that RIP is a novel form of apoptosis-inducing protein.
Recent years have seen a dramatic expansion in the range of applications of expression cloning techniques. New vectors and detection methods promise to further broaden the applicability of function-based screening approaches to problems in gene discovery. A major theme in the past year has been the introduction of engineered reporter cells that heighten the sensitivity with which clones expressing cDNAs can be identified.
Receptors which induce immune system effector function bear similar intracellular sequences and respond to aggregation through a nonreceptor tyrosine kinase-dependent pathway. The mechanism by which receptor aggregation leads to cell activation is poorly understood, but recent experiments with chimeric receptors and kinases have begun to simplify the analysis.
Protein kinases and phosphatases play an important role in signal transduction. In the T cell, activation via the T cell receptor-CD3 complex results in rapid tyrosine phosphorylation of proteins, as well as subsequent increases in serine/threonine phosphorylation. The Raf serine/threonine kinase has been implicated in many receptor signaling pathways, including those of platelet-derived growth factor, epidermal growth factor, insulin, and interleukin-2 receptors. We show here that Raf is associated with the T cell receptor-CD3 complex in unstimulated murine T cells. Using a COS cell expression system, we show that a hypophosphorylated form of Raf specifically associates with the CD3 gamma and delta chains but not with the CD3 epsilon or zeta chains. These results suggest that Raf mediates signal transduction from the gamma and delta chains of the T cell receptor-CD3 complex, analogous to the role played by ZAP-70 in signal transduction mediated by the zeta chain.
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Cell-surface receptors can be divided into three classes, depending on whether they transmit information by allosteric conformational change, by receptor dimerization, or by receptor aggregation. So far, only the first class of receptors has proven readily accessible to chemical techniques; this is no accident, but times are changing.
We describe the production and characterization of a novel monoclonal antibody (MAb) that recognizes a human endothelial cell antigen expressed mainly in inflamed and malignant disease states. We have used immunohistochemistry to determine the spectrum of reactivity of this MAb compared with that of a MAb to factor VIII-related antigen (MAb FVIII). MAb 4A11 does not react with several myeloid or lymphoid cell lines or with peripheral blood cells. Unlike MAb FVIII, MAb 4A11 does not react with platelets. MAb 4A11 reacts with most vascular endothelial cells in lymphoid tissue but with few (< 10%) endothelial cells in thymus, spleen, liver, lung, adrenal gland, placenta, testes, and skin. MAb 4A11 detects endothelial cells in diseased tissues such as rheumatoid and osteoarthritic synovium and psoriatic skin. Vascular endothelial cells in both adrenal tumors and cutaneous Kaposi's sarcomas lesions are MAb 4A11 reactive. In vitro the 4A11 antigen is not detectable on cultured human umbilical vein endothelial cells and its expression is not induced on these cells by treatment with lipopolysaccharide, interferon-gamma, interleukin-1 and -6, or tumor necrosis factor-alpha. However, in an in vivo model of allergic contact dermatitis the 4A11 antigen is upregulated differentially from other endothelial markers such as E-selectin, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1. In this dermal model of inflammation, poison ivy extract is applied to the skin and biopsies taken at 0, 6, and 24 hours. In addition to focal keratinocyte expression, 4A11 antigen is found on 11% of dermal endothelial cells at time 0 and antigen expression increases with time until 24 hours, when 4A11 antigen is present on 63% of the endothelial cells. Using thin layer chromatography, MAb 4A11 reacts with the H-5-2 [Fuc alpha 2Gal beta 4GlcNAc beta 3Gal beta 4Glc beta 1Cer] and Lewis(y)-6 [Fuc alpha 2Gal beta 4(Fuc alpha 3)GlcNAc beta 3Gal beta 4-Glc beta 1Cer] blood group glycolipids. The presence of the novel 4A11 antigen in inflamed and malignant tissues containing many blood vessels and its differential upregulation in allergic contact dermatitis may signify an important function for this antigen in the inflammatory process.
Many cellular recognition events in the immune system are initiated by aggregation of cell surface receptors that lack intrinsic protein-tyrosine kinase activity. Receptor-associated kinases related to the src protooncogene product have been found to be essential for cellular activation and may interact with the cytoplasmic domains of the antigen receptor chains. We show here that anti-CD16 antibody-mediated clustering of chimeric transmembrane proteins bearing a CD16 extracellular domain and a Src family kinase intracellular domain is not sufficient to initiate a cellular activation signal in T cells, whereas clustering of similar chimeras bearing Syk or ZAP-70 kinase sequences triggers calcium mobilization. Aggregation of the Syk chimera alone, or coaggregation of chimeras bearing Fyn and ZAP-70 kinases, suffices to initiate cytolytic effector function. The pattern of tyrosine phosphorylation induced by clustering of the Syk chimera is similar to the pattern induced by aggregation of T cell receptor.
The CD4 cell surface antigen belongs to the immunoglobulin superfamily and is the primary receptor for the human immunodeficiency virus 1 (HIV-1). The high affinity interaction between HIV-1 and CD4 is mediated by the viral envelope glycoprotein gp120. Recombinant soluble CD4 (rsCD4) has been shown in vitro to be an effective inhibitor of HIV-1 and HIV-2 propagation in lymphoid cells. A variety of antibody-like molecules were constructed, consisting of different parts of the extracellular domain of CD4 fused to immunoglobulin constant regions. The fusion proteins were expressed in mammalian cell lines and purified via affinity chromatography. The specificity and anti-viral effects of the different CD4-immunoglobulin constructs against HIV were analysed by different immunological tests, i.e., immunofluorescence, neutralisation and in vitro assays. In pharmacokinetic studies, differences were found in serum half-life between the four- and two-domain CD4 constructs in cynomolgus monkeys and between glycosylated and deglycosylated CD4-Fc constructs in rabbits. In two in vivo experiments using the four-domain CD4-Fc in SIV-infected macaques, no beneficial effects were observed.
A cDNA encoding the T cell activation Ag CD26 was isolated from human PHA-activated T cells by using an expression cloning method. The nucleotide sequence obtained predicts a protein of 766 amino acids of type II membrane topology, with six amino acids in the cytoplasmic region. The predicted amino acid sequence of the Ag was 85% homologous to that of the dipeptidyl peptidase IV enzyme isolated from rat liver. Derivatives of the human leukemic T cell line Jurkat transfected with a CD26 expression plasmid were established. Characterization of the CD26 Ag expressed by the transfected Jurkat cells revealed that the Ag could be immunoprecipitated as a 110-kDa molecule similar to that found on peripheral blood T cells and that the Ag had dipeptidyl peptidase IV activity. Functional analysis of these Jurkat transfectants showed that cross-linking of the CD26 and CD3 Ag with their respective antibodies resulted in enhanced intracellular calcium mobilization and IL-2 production. These results provide direct evidence that the CD26 Ag plays a role in T cell activation.
The zeta chain of the T cell antigen receptor is a dimeric transmembrane protein with a very short extracellular domain and an extended cytoplasmic tail that triggers T cell effector function when aggregated by extracellular stimuli. We have reduced the active site of zeta to an 18 residue motif that can be appended to the intracellular domain of other transmembrane proteins to endow them with receptor-like activity. The compact size of the motif appears to eliminate zeta mechanisms based on enzymatic activity and suggests that one or at most a few cellular proteins interact with the zeta intracellular domain to initiate signal transduction. Analysis of individual amino acids within the 18 residue element reveals two phylogenetically conserved tyrosines that are absolutely required for activity and other residues that are less essential but contribute to the efficacy of receptor-directed cytolysis.