Biomedical subjects

Tobias Zech

Publications and source records attributed to Tobias Zech.

4 recordsLinked to original sources

PubMed2004

Recognition sequences for the GYF domain reveal a possible spliceosomal function of CD2BP2.

Protein-protein interactions are often mediated by small domains that recognize solvent-exposed peptide sequences. Deciphering the recognition code for these adapter domains is an important step in the understanding of multi-protein assemblies. Here, we investigate the sequence requirements for the CD2BP2-GYF domain, a proline-rich sequence binding module previously shown to be involved in T cell signaling. We show that the signature (R/K/G)XXPPGX(R/K) defines a preferred peptide-binding motif that is present in several proteins of the splicing machinery. Specifically, the core small nuclear ribonucleoprotein, SmB/B', contains several PPP-PGMR motifs that interact with the CD2BP2-GYF domain in vitro and in vivo. The colocalization of CD2BP2 and SmB proteins in the nucleus of Jurkat T cells and HeLa cells suggests a function of the GYF domain of CD2BP2 in mediating protein-protein interactions within the spliceosome.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Biochemical and functional analysis of smallpox growth factor (SPGF) and anti-SPGF monoclonal antibodies.

Variola, the causative agent of smallpox, is a highly infectious double-stranded DNA virus of the orthopox genus that replicates within the cytoplasm of infected cells. For unknown reasons prominent skin manifestations, including "pox," mark the course of this systemic human disease. Here we characterized smallpox growth factor (SPGF), a protein containing an epidermal growth factor (EGF)-like domain that is conserved among orthopox viral genomes, and investigated its possible mechanistic link. We show that after recombinant expression, refolding, and purification, the EGF domain of SPGF binds exclusively to the broadly expressed cellular receptor, erb-B1 (EGF receptor), with subnanomolar affinity, stimulating the growth of primary human keratinocytes and fibroblasts. High affinity monoclonal antibodies specific for SPGF reveal in vivo immunoprotection in a murine vaccinia pneumonia model by a mechanism distinct from viral neutralization. These findings suggest that blockade of pathogenic factor actions, in general, may be advantageous to the infected host.

Amino Acid Sequence↗

PubMed2003

Synergistic assembly of linker for activation of T cells signaling protein complexes in T cell plasma membrane domains.

Transmembrane adaptor molecule LAT (linker for activation of T cells) forms a central scaffold for signaling protein complexes that accumulate in the vicinity of activated T cell antigen receptors (TCR). Here we used biochemical analysis of immunoisolated plasma membrane domains and fluorescence imaging of green fluorescence protein-tagged signaling proteins to investigate the contributions of different tyrosine-based signaling protein docking sites of LAT to the formation of LAT signaling protein assemblies in TCR membrane domains. We found that the phospholipase C gamma docking site of LAT and different Grb2/Gads docking sites function in an interdependent fashion and synergize to accumulate LAT, Grb2, and phospholipase C gamma in TCR signaling assemblies. Two-dimensional gels showed that Grb2 is a predominant cytoplasmic adaptor in the isolated LAT signaling complexes, whereas Gads, Crk-1, and Grap are present in lower amounts. Taken together our data suggest a synergistic assembly of multimolecular TCR.LAT signal transduction complexes in T cell plasma membrane domains.

Adaptor Proteins, Signal Transducing↗

PubMedDate not supplied

Visualizing membrane microdomains by Laurdan 2-photon microscopy.

Lateral segregation of cell membrane components gives rise to microdomains with a different structure within the membrane. Most prominently, lipid rafts are defined as domains in liquid ordered phase whereas surrounding membranes are more fluid. Here we review a 2-photon fluorescence microscopy approach, which allows the visualization of membrane fluidity. The fluorescent probe Laurdan exhibits a blue shift in emission with increasing membrane condensation caused by an alteration in the dipole moment of the probe as a consequence of exclusion of water molecules from the lipid bilayer. The quantification of membrane order is achieved by the Generalized Polarization (GP) values, which are defined as normalized intensity ratios of two emission channels. GP images are therefore not biased by probe concentrations and membrane ruffles. Furthermore, Laurdan reports membrane structure independently from the lipid and protein cargo of the membrane domains. We give examples where Laurdan microscopy was instrumental in quantifying the formation of condensed membrane domains and their cellular requirements. Moreover we discuss how microdomains identified by Laurdan microscopy are consistent with domains identified by other methodologies and put GP images in the context of current raft hypotheses.

2-Naphthylamine↗