The transmembrane adapter protein SIT regulates thymic development and peripheral T-cell functions

SIT is a transmembrane adapter protein that modulates signals emanating from the T-cell receptor (TCR). Here, we have used gene-targeted mice to assess the role of SIT for T-cell development and peripheral T-cell functions. SIT^−/− double-positive thymocytes show an upregulation of the activation markers CD5 and CD69, suggesting that SIT negatively regulates TCR-mediated signals at the CD4⁺ CD8⁺ stage of thymic development. This assumption is further supported by the observation that in female H-Y TCR transgenic mice, positive selection is enhanced and even converted to negative selection. Similarly, mature peripheral T cells are hyperresponsive towards TCR-mediated stimuli and produce larger amounts of T-helper 1 (TH1) cytokines, and SIT-deficient mice show an increased susceptibility to develop experimental autoimmune encephalomyelitis, a mouse model of multiple sclerosis. These results demonstrate that SIT is a critical negative regulator of TCR-mediated signaling and finely tunes the signals required for thymic selection and peripheral T-cell activation.     

The Salmonella pathogenicity island (SPI)-2 and SPI-1 type III secretion systems allow Salmonella serovar typhimurium to trigger colitis via MyD88-dependent and MyD88-independent mechanisms

Salmonella typhimurium can colonize the gut, invade intestinal tissues, and cause enterocolitis. In vitro studies suggest different mechanisms leading to mucosal inflammation, including 1) direct modulation of proinflammatory signaling by bacterial type III effector proteins and 2) disruption or penetration of the intestinal epithelium so that penetrating bacteria or bacterial products can trigger innate immunity (i.e., TLR signaling). We studied these mechanisms in vivo using streptomycin-pretreated wild-type and knockout mice including MyD88(-/-) animals lacking an adaptor molecule required for signaling via most TLRs. The Salmonella SPI-1 and the SPI-2 type III secretion systems (TTSS) contributed to inflammation. Mutants that retain only a functional SPI-1 (M556; sseD::aphT) or a SPI-2 TTSS (SB161; DeltainvG) caused attenuated colitis, which reflected distinct aspects of the colitis caused by wild-type S. typhimurium: M556 caused diffuse cecal inflammation that did not require MyD88 signaling. In contrast, SB161 induced focal mucosal inflammation requiring MyD88. M556 but not SB161 was found in intestinal epithelial cells. In the lamina propria, M556 and SB161 appeared to reside in different leukocyte cell populations as indicated by differential CD11c staining. Only the SPI-2-dependent inflammatory pathway required aroA-dependent intracellular growth. Thus, S. typhimurium can use two independent mechanisms to elicit colitis in vivo: SPI-1-dependent and MyD88-independent signaling to epithelial cells and SPI-2-dependent intracellular proliferation in the lamina propria triggering MyD88-dependent innate immune responses.     

Kaposi's sarcoma and human chorionic gonadotropin: mechanisms,moieties and mysteries

Kaposi's Sarcoma (KS) is a highly angiogenic neoplasm associated with infection by the human gamma-herpesvirus, HHV-8 or Kaposi's sarcoma herpes virus (KSHV). When in 1872 the Hungarian scientist Moritz Kaposi described the sarcoma, which was later named after him, he was dealing with a rare dermatologic disease. Today, KS is a more common pathology due to its high incidence in AIDS, in immuno-suppressed transplantation patients and, in its endemic form, in Africa. The introduction of highly active antiretroviral therapy (HAART) has led to a drastic reduction of KS incidence in HIV-infected patients, but in some cases KS resists the treatment. KS is more common in men than in women. The observation of spontaneous remissions during pregnancy stimulated investigations into the potential anti-KS activity of the pregnancy hormone human chorionic gonadotropin (hCG). The variable effect in clinical trials using urinary preparations of the hormone (u-hCG) has led to the hypothesis that contaminating moieties present in these preparations may account for the anti-KS effect observed in vitro. While the discrepancy between laboratory tests and clinical trials remains a mystery, little is known about potential anti-KS mechanisms of the hormone itself and/or other active moieties present in u-hCG.     

Self-assembly is important for TIP47 function in mannose 6-phosphate receptor transport

TIP47 (tail-interacting protein of 47 kDa) binds to the cytoplasmic domains of mannose 6-phosphate receptors and is required for their transport from endosomes to the trans- Golgi network in vitro and in living cells. TIP47 occurs in cytosol as an oligomer; it chromatographs with an apparent mass of ∼ 300 kDa and displays an S -value of ∼ 13. Recombinant TIP47 forms homo-oligomers that are likely to represent hexamers, as determined by chemical cross-linking. Removal of TIP47 residues 1–151 yields a protein that behaves as a monomer upon gel filtration, yet is fully capable of binding mannose 6-phosphate receptor cytoplasmic domains. The presence of an oligomerization domain in the N-terminus of TIP47 was confirmed by expression of N-terminal residues 1–133 or 1–257 in mammalian cells. Co-expression of full-length TIP47 with either of these fragments led to the formation of higher-order aggregates of wild-type TIP47. Furthermore, the N-terminal domains expressed alone also occurred as oligomers. These studies reveal an N-terminal oligomerization domain in TIP47, and show that oligomerization is not required for TIP47 recognition of mannose 6-phosphate receptors. However, oligomerization is required for TIP47 stimulation of mannose 6-phosphate receptor transport from endosomes to the trans- Golgi in vivo .     

Synthesis and reactivity of early-late heterobimetallic complexes displaying a η-tantalum-alkylidene to palladium interaction

Tantalaalkylidene compounds, CHRTaCl₃L₂ (R=^tBu or CMe₂Ph, L=THF or 1/2dimethoxyethane) mixed with the cyclopalladated dimer [Pd(2-C₆H₄CH₂NMe₂)(μ-Cl)]₂, 1, afford good yields of heterodimetallic complexes [Pd(2-C₆H₄CH₂NMe₂)(μ-Cl)(μ-CHR=TaCl₃L], 3a, 3b, in which the TaC unit is η²-interacting with the palladium atom, while a chloride ligand is bridging the tantalum and the palladium atoms. These compounds are fairly stable in air in the solid state and also in solution at RT. The interaction of the TaC unit with Pd in these bimetallic compounds is weak as shown by the ready formation of [Pd(2-C₆H₄CH₂NMe₂)PyCl] and CHRTaCl₃Py₂ upon treatement with pyridine. Compounds analogous to 3a, b can also be obtained with 12 electrons tantalum complexes. Thus treating the same cyclopalladated dimer 1 with CHRTa(OAr)₃ (OAr=2,6-diisopropylphenyloxy) led to a much more stable though electron deficient species: [Pd(2-C₆H₄CH₂NMe₂)(μ-Cl)(μ-CH^tBu=Ta(OAr)₃], 3c. Substitution in 3a of one chloride ion by an alkyl group occurred at the tantalum metal via reaction with ZnR₂ (R=CH₂CMe₂Ph) leading to [Pd(2-C₆H₄CH₂NMe₂)(μ-Cl)(μ-CH^tBu=TaCl₃(CH₂CMe₂Ph)], 4 for which there is no free rotation around the new TaC bond and in which one of the methylene protons is strongly interacting with the palladium centre. This compound is believed to mimic an intermediate to the formation of tantalacarbyne derivative, which was obtained earlier via reaction of the uncomplexed tantalacarbene compound with dialkylzinc compounds.     

In vitro selection and prediction of TIP47 protein-interaction interfaces

We present a new method for the rapid identification of amino acid residues that contribute to protein-protein interfaces. Tail-interacting protein of 47 kDa (TIP47) binds Rab9 GTPase and the cytoplasmic domains of mannose 6-phosphate receptors and is required for their transport from endosomes to the Golgi apparatus. Cysteine mutations were incorporated randomly into TIP47 by expression in Escherichia coli cells harboring specific misincorporator tRNAs. We made use of the ability of the native TIP47 protein to protect 48 cysteine probes from chemical modification by iodoacetamide as a means to obtain a surface map of TIP47, revealing the identity of surface-localized, hydrophobic residues that are likely to participate in protein-protein interactions. Direct mutation of predicted interface residues confirmed that the protein had altered binding affinity for the mannose 6-phosphate receptor. TIP47 mutants with enhanced or diminished affinities were also selected by affinity chromatography. These methods were validated in comparison with the protein's crystal structure, and provide a powerful means to predict protein-protein interaction interfaces.     

Signal via lymphotoxin-beta R on bone marrow stromal cells is required for an early checkpoint of NK cell development

NK cells play an important role in the immune system but the cellular and molecular requirements for their early development are poorly understood. Lymphotoxin-alpha (LTalpha)(-/-) and LTbetaR(-/-) mice show a severe systemic reduction of NK cells, which provides an excellent model to study NK cell development. In this study, we show that the bone marrow (BM) or fetal liver cells from LTalpha(-/-) or LTbetaR(-/-) mice efficiently develop into mature NK cells in the presence of stromal cells from wild-type mice but not from LTalpha(-/-) or LTbetaR(-/-) mice. Direct activation of LTbetaR-expressing BM stromal cells is shown to promote to early NK cell development in vitro. Furthermore, the blockade of the interaction between LT and LTbetaR in adult wild-type mice by administration of LTbetaR-Ig impairs the development of NK cells in vivo. Together, these results indicate that the signal via LTbetaR on BM stromal cells by membrane LT is an important pathway for early NK cell development.     

Intact lysosome transport and phagosome function despite kinectin deficiency

The mechanism of cargo coupling to kinesin motor proteins is a fundamental issue in organelle transport along microtubules. Kinectin has been postulated to function as a membrane anchor protein that attaches various organelles to the prototype motor protein kinesin. To verify the biological relevance of kinectin in vivo, the murine kinectin gene was disrupted by homologous recombination. Unexpectedly, kinectin-deficient mice were viable and fertile, and no gross abnormalities were observed up to 1 year of age. The assembly of the endoplasmic reticulum was essentially unaffected in kinectin-deficient cells. Mitochondria appeared to be correctly distributed throughout the cytoplasm along the microtubules. Furthermore, the stationary distribution and the bidirectional movement of lysosomes did not depend on kinectin. Kinectin-deficient phagocytes internalized and cleared bacteria, indicating that phagosome trafficking and maturation are functional without kinectin. Thus, these data unequivocally indicate that kinectin is not essential for trafficking of lysosomes, phagosomes, and mitochondria in vivo.