ABSTRACT
The pleiotropic cytokine transforming growth factor-ß (TGF-ß) signals through different pathways among which the Smad- and the MAP-Kinase pathways are already well characterized. Both pathways utilize adaptor/chaperone molecules that facilitate or modulate the intracellular signaling events. Two of the proteins shown in vitro to play a role in Smad-dependent signaling are the TGF-ß Receptor Associated Protein-1 (TRAP1, also TGFBRAP1) and its homologue VPS39, also known as Vam6 and TRAP1-Like-Protein (TLP). We generated mice deficient for TRAP1 and VPS39/TLP, respectively. Absence of TRAP1 protein results in death at either of two defined timepoints during embryogenesis, before the blastula stage or during gastrulation, whereas most of the VPS39 deficient mice die before E6.5. Heterozygous mice show no overt phenotype. In summary, our data indicate that TRAP1 and VPS39 are nonredundant and essentially required for early embryonic development.
Subject(s)
Blastula/embryology , Embryonic Development , Gastrula/embryology , Guanine Nucleotide Exchange Factors/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Transforming Growth Factor beta/metabolism , Animals , Animals, Genetically Modified , Autophagy-Related Proteins , Blotting, Northern , Blotting, Western , Cells, Cultured , Gene Expression , Genotype , Guanine Nucleotide Exchange Factors/genetics , HSP90 Heat-Shock Proteins , Intracellular Signaling Peptides and Proteins/genetics , Mice , Polymerase Chain Reaction , Signal Transduction , Vesicular Transport ProteinsABSTRACT
IFN-gamma orchestrates a potent antimicrobial host response. However, the underlying molecular basis for this immunological defense system is largely unknown. In a systematic approach to identify IFN-gamma-regulated host effector molecules, a notable number of transcripts with consensus GTP-binding motives were obtained. Further extensive transcriptome and genome analyses identified five novel family members of murine guanylate-binding proteins (mGBPs) now designated mGBP6, 7, 8, 9, and 10. Moreover, in this study, all 10 mGBP members (mGBP1-10) were extensively characterized. mGBPs are selectively up-regulated in vitro by a set of proinflammatory cytokines and TLR agonists as well as in vivo after Listeria monocytogenes and Toxoplasma gondii infection. After IFN-gamma stimulation, mGBP1, 2, 3, 6, 7, and 9 are associated with intracellular Toxoplasma parasites and, interestingly, virulent Toxoplasma interfere with mGBP recruitment. Taken together, mGBPs comprise an important set of host defense molecules.
Subject(s)
GTP-Binding Proteins/drug effects , GTP-Binding Proteins/metabolism , Interferon-gamma/pharmacology , Amino Acid Sequence , Animals , Cell Line , Cells, Cultured , Disease Models, Animal , GTP-Binding Proteins/genetics , GTP-Binding Proteins/immunology , Injections, Intraperitoneal , Listeria monocytogenes/immunology , Listeria monocytogenes/isolation & purification , Listeriosis/immunology , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Toxoplasma/immunology , Toxoplasma/isolation & purification , Toxoplasmosis, Animal/immunology , Up-Regulation/immunologyABSTRACT
In a systematic approach to identify interferon-gamma (IFN-gamma)-regulated host effector molecules, we found several members of the 65 kDa guanylate-binding proteins (GBPs) highly upregulated. During extensive characterization of these guanosine triphosphatases (GTPases), we identified discrepancies between the cloned and published sequences of the murine GTPase mGBP4. Two splice variants of mGBP4 could be detected. One variant led to a premature stop codon after 312 bp. The second variant resulted in a transcript with a disrupted G2 domain and was deposited as mGBP4.1 to the GenBank. Interestingly, only mGBP4, not mGBP4.1 mRNA, was highly upregulated in mice after infection with Listeria monocytogenes.
