The ADAP/SKAP55 signaling module regulates T-cell receptor-mediated integrin activation through plasma membrane targeting of Rap1

Adhesion of T cells after stimulation of the T-cell receptor (TCR) is mediated via signaling processes that have collectively been termed inside-out signaling. The molecular basis for inside-out signaling is not yet completely understood. Here, we show that a signaling module comprising the cytosolic adapter proteins ADAP and SKAP55 is involved in TCR-mediated inside-out signaling and, moreover, that the interaction between ADAP and SKAP55 is mandatory for integrin activation. Disruption of the ADAP/SKAP55 module leads to displacement of the small GTPase Rap1 from the plasma membrane without influencing its GTPase activity. These findings suggest that the ADAP/SKAP55 complex serves to recruit activated Rap1 to the plasma membrane. In line with this hypothesis is the finding that membrane targeting of the ADAP/SKAP55 module induces T-cell adhesion in the absence of TCR-mediated stimuli. However, it appears as if the ADAP/SKAP55 module can exert its signaling function outside of the classical raft fraction of the cell membrane.     

Normal T-cell development and immune functions in TRIM-deficient mice

The transmembrane adaptor molecule TRIM is strongly expressed within thymus and in peripheral CD4(+) T cells. Previous studies suggested that TRIM is an integral component of the T-cell receptor (TCR)/CD3 complex and might be involved in regulating TCR cycling. To elucidate the in vivo function of TRIM, we generated TRIM-deficient mice by homologous recombination. TRIM(-/-) mice develop normally and are healthy and fertile. However, the animals show a mild reduction in body weight that appears to be due to a decrease in the size and/or cellularity of many organs. The morphology and anatomy of nonlymphoid as well as primary and secondary lymphoid organs is normal. The frequency of thymocyte and peripheral T-cell subsets does not differ from control littermates. In addition, a detailed analysis of lymphocyte development revealed that TRIM is not required for either positive or negative selection. Although TRIM(-/-) CD4(+) T cells showed an augmented phosphorylation of the serine/threonine kinase Akt, the in vitro characterization of peripheral T cells indicated that proliferation, survival, activation-induced cell death, migration, adhesion, TCR internalization and recycling, TCR-mediated calcium fluxes, tyrosine phosphorylation, and mitogen-activated protein family kinase activation are not affected in the absence of TRIM. Similarly, the in vivo immune response to T-dependent and T-independent antigens as well as the clinical course of experimental autoimmune encephalomyelitis, a complex Th1-mediated autoimmune model, is comparable to that of wild-type animals. Collectively, these results demonstrate that TRIM is dispensable for T-cell development and peripheral immune functions. The lack of an evident phenotype could indicate that TRIM shares redundant functions with other transmembrane adaptors involved in regulating the immune response.     

Signaling signatures and functional properties of anti-human CD28 superagonistic antibodies

Superagonistic CD28 antibodies (CD28SAs) activate T lymphocytes without concomitant perturbation of the TCR/CD3-complex. In rodents these reagents induce the preferential expansion of regulatory T cells and can be used for the treatment of autoimmune diseases. Unexpectedly, the humanized CD28 superagonist TGN1412 caused severe and life threatening adverse effects during a recently conducted phase I clinical trail. The underlying molecular mechanisms are as yet unclear. We show that TGN1412 as well as the commercially available CD28 superagonist ANC28.1 induce a delayed but extremely sustained calcium response in human naïve and memory CD4⁺ T cells but not in cynomolgus T lymphocytes. The sustained Ca⁺⁺-signal was associated with the activation of multiple intracellular signaling pathways and together these events culminated in the rapid de novo synthesis of high amounts of pro-inflammatory cytokines, most notably IFN-γ and TNF-α. Importantly, sustained transmembranous calcium flux, activation of Src-kinases as well as activation of PI3K were found to be absolutely required for CD28SA-mediated production of IFN-γ and IL-2. Collectively, our data suggest a molecular basis for the severe side effects caused by TGN1412 and impinge upon the relevance of non-human primates as preclinical models for reagents that are supposed to modify the function of human T cells.     

Thiol-specific cross-linkers of variable length reveal a similar separation of SH1 and SH2 in myosin subfragment 1 in the presence and absence of MgADP.

A series of thiol-specific cross-linking reagents were prepared for studying the kinetics of cross-linking between SH1 (Cys(707)) and SH2 (Cys(697)) in rabbit skeletal muscle myosin subfragment 1. The reagents were of the type RSS(CH(2))(n)()SSR, with R = 3-carboxy-4-nitrophenyl and n = 3, 6, 7, 8, 9, 10, and 12, spanning distances from 9 to 20 A. The reactions were monitored spectrophotometrically by measuring the release of 2-nitro-5-thiobenzoate. Reaction rates for modification of SH1 (k(1)) and for cross-linking (k(2)) were measured by the decrease of the K(+)(EDTA)-ATPase activity and the decrease of the Ca(2+)-ATPase activity, respectively, and corrected for the different reactivities of C(n). Cross-linking rates in the presence and absence of MgADP showed similar dependence on the length of the reagents: While the cross-linking rates for n = 3 or n = 6 were close to those for n = 0 (Ellman's reagent), those for n = 7 and 8 were significantly increased. Thus the distance between SH1 and SH2 appears to be equal in both states and can be estimated as >/=15 A, based on the length of the reagent with n = 8 in stretched conformation. Under rigor conditions, reactivity of SH1 differed significantly from that in the presence of MgADP, presumably because of shielding through a lipophilic domain. Similarly, the cross-linking rates k(2) for C(3), C(6), and C(7) in the absence of MgADP were ca. 15 times lower than in the presence of MgADP, suggesting a change in the structure of the SH2 region that depends on nucleotide binding. The results are discussed in terms of recent X-ray structures of S1 and S1-MgADP [Rayment et al. (1993) Science 261, 50-58; Gulick et al. (1997) Biochemistry 36, 11619-11628].     

Screening for proteolytically active lactic acid bacteria and bioactivity of peptide hydrolysates obtained with selected strains

Applied Microbiology and Biotechnology - In a screening for proteolytically active lactic acid bacteria, three strains, Lactobacillus delbrueckii ssp. lactis 92202, Lactobacillus helveticus 92201,...     

A Mammalian Cell Based FACS-Panning Platform for the Selection of HIV-1 Envelopes for Vaccine Development

An increasing number of broadly neutralizing monoclonal antibodies (bnMAb) against the HIV-1 envelope (Env) protein has been discovered recently. Despite this progress, vaccination efforts with the aim to re-elicit bnMAbs that provide protective immunity have failed so far. Herein, we describe the development of a mammalian cell based FACS-panning method in which bnMAbs are used as tools to select surface-exposed envelope variants according to their binding affinity. For that purpose, an HIV-1 derived lentiviral vector was developed to infect HEK293T cells at low multiplicity of infection (MOI) in order to link Env phenotype and genotype. For proof of principle, a gp145 Env model-library was established in which the complete V3 domain was substituted by five strain specific V3 loop sequences with known binding affinities to nMAb 447-52D, respectively. Env genes were recovered from selected cells by PCR, subcloned into a lentiviral vector (i) to determine and quantify the enrichment nMAb binders and (ii) to generate a new batch of transduction competent particles. After 2 selection cycles the Env variant with highest affinity was enriched 20-fold and represented 80% of the remaining Env population. Exploiting the recently described bnMAbs, this procedure might prove useful in selecting Env proteins from large Env libraries with the potential to elicit bnMAbs when used as vaccine candidates.     

Characterization of a Borna disease virus glycoprotein, gp18.

Borna disease virus is a nonsegmented negative-strand RNA virus that causes neurologic disease in a wide variety of animal hosts. Here we describe identification and characterization of the first glycoprotein in this viral system. The 18-kDa glycoprotein, gp18, has been purified from infected rat brain. Isolation and microsequencing of this protein allowed identification of a 16.2-kDa open reading frame in the viral antigenome. Lectin binding and endoglycosidase sensitivity assays indicate that gp18 is an unusual N-linked glycoprotein.     

p38 MAPK inhibition is critically involved in VEGFR-2-mediated endothelial cell survival

Vascular endothelial growth factor (VEGF) promotes vasculogenesis, arteriogenesis, and angiogenesis by stimulating proliferation, migration, and cell survival of endothelial cells. VEGF mediates its actions through activation of two receptor tyrosine kinases, VEGFR-1 and VEGFR-2. Serum starvation led to apoptosis of human umbilical vein endothelial cells (HUVEC), which was accompanied by activation of p38 MAPK and caspase-3. Stimulation of both VEGF-receptors resulted in a considerable decrease of apoptosis, which was associated with the inhibition of p38 MAPK and caspase-3 activity. Selective stimulation of VEGFR-2 showed similar results, whereas the isolated activation of VEGFR-1 was without effect. Incubation of HUVEC with SB203580, a p38 MAPK inhibitor, resulted in similar effects as VEGF-stimulation: p38 MAPK and caspase-3 enzyme activity were reduced and apoptosis was prevented. These data indicate that activation of VEGFR-2 prevents endothelial cell apoptosis by inhibiting p38 MAPK phosphorylation and thus, reducing caspase-3 activity.     

Embedding permanent watermarks in synthetic genes

As synthetic biology advances, labeling of genes or organisms, like other high-value products, will become important not only to pinpoint their identity, origin, or spread, but also for intellectual property, classification, bio-security or legal reasons. Ideally information should be inseparably interlaced into expressed genes. We describe a method for embedding messages within open reading frames of synthetic genes by adapting steganographic algorithms typically used for watermarking digital media files. Text messages are first translated into a binary string, and then represented in the reading frame by synonymous codon choice. To aim for good expression of the labeled gene in its host as well as retain a high degree of codon assignment flexibility for gene optimization, codon usage tables of the target organism are taken into account. Preferably amino acids with 4 or 6 synonymous codons are used to comprise binary digits. Several different messages were embedded into open reading frames of T7 RNA polymerase, GFP, human EMG1 and HIV gag, variously optimized for bacterial, yeast, mammalian or plant expression, without affecting their protein expression or function. We also introduced Vigenère polyalphabetic substitution to cipher text messages, and developed an identifier as a key to deciphering codon usage ranking stored for a specific organism within a sequence of 35 nucleotides.