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1.
bioRxiv ; 2024 Jun 25.
Article in English | MEDLINE | ID: mdl-38562771

ABSTRACT

Legionella pneumophila grows within membrane-bound vacuoles in phylogenetically diverse hosts. Intracellular growth requires the function of the Icm/Dot type-IVb secretion system, which translocates more than 300 proteins into host cells. A screen was performed to identify L. pneumophila proteins that stimulate MAPK activation, using Icm/Dot translocated proteins ectopically expressed in mammalian cells. In parallel, a second screen was performed to identify L. pneumophila proteins expressed in yeast that cause growth inhibition in MAPK pathway-stimulatory high osmolarity medium. LegA7 was shared in both screens, a protein predicted to be a member of the bacterial cysteine protease family that has five carboxyl-terminal ankyrin repeats. Three conserved residues in the predicted catalytic triad of LegA7 were mutated. These mutations abolished the ability of LegA7 to inhibit yeast growth. To identify other residues important for LegA7 function, a generalizable selection strategy in yeast was devised to isolate mutants that have lost function and no longer cause growth inhibition on high osmolarity medium. Mutations were isolated in the two carboxyl-terminal ankyrin repeats, as well as an inter-domain region located between the cysteine protease domain and the ankyrin repeats. These mutations were predicted by AlphaFold modeling to localize to the face opposite from the catalytic site, arguing that they interfere with the positive regulation of the catalytic activity. Based on our data, we present a model in which LegA7 harbors a cysteine protease domain with an inter-domain and two carboxyl-terminal ankyrin repeat regions that modulate the function of the catalytic domain.

2.
Cell Microbiol ; 11(2): 230-48, 2009 Feb.
Article in English | MEDLINE | ID: mdl-19016775

ABSTRACT

The bacterial pathogen Legionella pneumophila replicates in a specialized vacuole within host cells. Establishment of the replication vacuole depends on the Dot/Icm translocation system that delivers a large number of protein substrates into the host cell. The functions of most substrates are unknown. Here, we analysed a defined set of 127 confirmed or candidate Dot/Icm substrates for their effect on host cell processes using yeast as a model system. Expression of 79 candidates caused significant yeast growth defects, indicating that these proteins impact essential host cell pathways. Notably, a group of 21 candidates interfered with the trafficking of secretory proteins to the yeast vacuole. Three candidates that caused yeast secretory defects (SetA, Ceg19 and Ceg9) were investigated further. These proteins impinged upon vesicle trafficking at distinct stages and had signals that allowed translocation into host cells by the Dot/Icm system. Ectopically produced SetA, Ceg19 and Ceg9 localized to secretory organelles in mammalian cells, consistent with a role for these proteins in modulating host cell vesicle trafficking. Interestingly, the ability of SetA to cause yeast phenotypes was dependent upon a functional glycosyltransferase domain. We hypothesize that SetA may glycosylate a component of the host cell vesicle trafficking machinery during L. pneumophila infection.


Subject(s)
Bacterial Proteins/metabolism , Legionella pneumophila/physiology , Saccharomyces cerevisiae/growth & development , Virulence Factors/metabolism , Amino Acid Sequence , Animals , Bacterial Proteins/genetics , Cell Line , Endoplasmic Reticulum/metabolism , Epithelial Cells/microbiology , Golgi Apparatus/metabolism , Humans , Macrophages/microbiology , Mice , Molecular Sequence Data , Saccharomyces cerevisiae/genetics , Transport Vesicles/metabolism , Virulence Factors/genetics
3.
Proc Natl Acad Sci U S A ; 102(52): 19097-102, 2005 Dec 27.
Article in English | MEDLINE | ID: mdl-16365311

ABSTRACT

The origin of the pathogenic endothelial cells in common infantile hemangioma is unknown. We show here that the transcriptomes of human placenta and infantile hemangioma are sufficiently similar to suggest a placental origin for this tumor, expanding on recent immunophenotypical studies that have suggested this possibility [North, P. E., et al. (2001) Arch. Dermatol. 137, 559-570]. The transcriptomes of placenta, hemangioma, and eight normal and diseased tissues were compared by hierarchical and nonhierarchical clustering analysis of >7,800 genes. We found that the level of transcriptome similarity between placenta and hemangioma exceeded that of any other tissue compared and paralleled that observed between a given tissue and its derived tumor, such as normal and cancerous lung. The degree of similarity was even greater when a subset of endothelial cell-specific genes was analyzed. Genes preferentially expressed in both placenta and hemangiomas were identified, including 17-beta hydroxysteroid dehydrogenase type 2 and tissue factor pathway inhibitor 2. These data demonstrate the value of global molecular profiling of tissues as a tool for hypothesis-driven research. Furthermore, it suggests that the unique self-limited growth of infantile hemangioma may, in fact, mirror the lifetime of placental endothelium.


Subject(s)
Gene Expression Regulation, Neoplastic , Hemangioma/genetics , Hemangioma/pathology , Placenta/pathology , Vascular Neoplasms/genetics , Vascular Neoplasms/pathology , 17-Hydroxysteroid Dehydrogenases/genetics , Cluster Analysis , Databases, Genetic , Endothelium, Vascular/pathology , Gene Expression Regulation , Glycoproteins/genetics , Hemangioma/metabolism , Humans , Immunohistochemistry , Immunophenotyping , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Placenta/metabolism , RNA/metabolism , RNA, Messenger/metabolism , Tissue Distribution , Vascular Neoplasms/metabolism
4.
Proc Natl Acad Sci U S A ; 101(32): 11658-63, 2004 Aug 10.
Article in English | MEDLINE | ID: mdl-15289610

ABSTRACT

Spatio-temporal studies on the growth of capillary blood vessels and capillary lymphatic vessels in tissue remodeling have suggested that lymphangiogenesis is angiogenesis-dependent. We revisited this concept by using fibroblast growth factor 2 (FGF-2) (80 ng) to stimulate the growth of both vessel types in the mouse cornea. When we lowered the dose of FGF-2 in the cornea 6.4-fold (12.5 ng), the primary response was lymphangiogenic. Further investigation revealed that vascular endothelial growth factor-C and -D are required for this apparent lymphangiogenic property of FGF-2, and when the small amount of accompanying angiogenesis was completely suppressed, lymphangiogenesis remained unaffected. Our findings demonstrate that there is a dose-dependent response of FGF-2 for lymphangiogenesis, and lymphangiogenesis can occur in the absence of a preexisting or developing vascular bed, i.e., in the absence of angiogenesis, in the mouse cornea.


Subject(s)
Fibroblast Growth Factor 2/pharmacology , Lymphangiogenesis/drug effects , Animals , Cell Division/drug effects , Cell Movement/drug effects , Cornea/blood supply , Cornea/physiology , Corneal Neovascularization , Dose-Response Relationship, Drug , Endothelial Cells/cytology , Endothelial Cells/drug effects , Male , Mice , Mice, Inbred Strains , Neovascularization, Physiologic/drug effects , Vascular Endothelial Growth Factor C/pharmacology , Vascular Endothelial Growth Factor D/pharmacology
5.
J Clin Invest ; 110(7): 923-32, 2002 Oct.
Article in English | MEDLINE | ID: mdl-12370270

ABSTRACT

Several drugs approved for a variety of indications have been shown to exhibit antiangiogenic effects. Our study focuses on the PPARgamma ligand rosiglitazone, a compound widely used in the treatment of type 2 diabetes. We demonstrate, for the first time to our knowledge, that PPARgamma is highly expressed in tumor endothelium and is activated by rosiglitazone in cultured endothelial cells. Furthermore, we show that rosiglitazone suppresses primary tumor growth and metastasis by both direct and indirect antiangiogenic effects. Rosiglitazone inhibits bovine capillary endothelial cell but not tumor cell proliferation at low doses in vitro and decreases VEGF production by tumor cells. In our in vivo studies, rosiglitazone suppresses angiogenesis in the chick chorioallantoic membrane, in the avascular cornea, and in a variety of primary tumors. These results suggest that PPARgamma ligands may be useful in treating angiogenic diseases such as cancer by inhibiting angiogenesis.


Subject(s)
Angiogenesis Inhibitors/pharmacology , Antineoplastic Agents/pharmacology , Neoplasm Metastasis/prevention & control , Receptors, Cytoplasmic and Nuclear/physiology , Thiazoles/pharmacology , Thiazolidinediones , Transcription Factors/physiology , Animals , Cattle , Fibroblast Growth Factor 2/physiology , Humans , Ligands , Neoplasm Invasiveness
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