ABSTRACT
Type III secretion systems (TTSSs) utilized by enteropathogenic bacteria require the presence of small, acidic virulence-associated chaperones for effective host cell infection. We adopted a combination of biochemical and cellular techniques to define the chaperone binding domains (CBDs) in the translocators IpaB and IpaC associated with the chaperone IpgC from Shigella flexneri. We identified a novel CBD in IpaB and furthermore precisely mapped the boundaries of the CBDs in both translocator proteins. In IpaC a single binding domain associates with IpgC. In IpaB, we show that the binding of the newly characterized CBD is essential in maintaining the ternary arrangement of chaperone-translocator complex. This hitherto unknown function is reflected in the co-crystal structure as well, with an IpgC dimer bound to an IpaB fragment comprising both CBDs. Moreover, in the absence of this novel CBD the IpaB/IpgC complex aggregates. This dual-recognition of a domain in the protein by the chaperone in facilitating the correct chaperone-substrate organization describes a new function for the TTSS associated chaperone-substrate complexes.
Subject(s)
Antigens, Bacterial/metabolism , Bacterial Proteins/metabolism , Bacterial Secretion Systems/physiology , Molecular Chaperones/metabolism , Multiprotein Complexes/metabolism , Shigella flexneri/metabolism , Antigens, Bacterial/chemistry , Antigens, Bacterial/genetics , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Crystallography, X-Ray , Molecular Chaperones/chemistry , Molecular Chaperones/genetics , Multiprotein Complexes/chemistry , Multiprotein Complexes/genetics , Operon/physiology , Peptide Mapping/methods , Protein Binding , Protein Multimerization/physiology , Protein Structure, Quaternary , Protein Structure, Tertiary , Shigella flexneri/chemistry , Shigella flexneri/geneticsABSTRACT
The angiopoietin/Tie2 system has been identified as the second vascular-specific receptor tyrosine kinase system controlling vessel assembly, maturation, and quiescence. Angiopoietin-2 (Ang-2) is prominently up-regulated in the host-derived vasculature of most tumors, making it an attractive candidate for antiangiogenic intervention. Yet, the net outcome of Ang-2 functions on tumor angiogenesis is believed to be contextual depending on the local cytokine milieu. Correspondingly, Ang-2 manipulatory therapies have been shown to exert protumorigenic as well as antitumorigenic effects. To clarify the role of Ang-2 for angiogenesis and tumor growth in a definite genetic experimental setting, the present study was aimed at comparatively studying the growth of different tumors in wild-type and Ang-2-deficient mice. Lewis lung carcinomas, MT-ret melanomas, and B16F10 melanomas all grew slower in Ang-2-deficient mice. Yet, tumor growth in wild-type and Ang-2-deficient mice dissociated during early stages of tumor development, whereas tumor growth rates during later stages of primary tumor progression were similar. Analysis of the intratumoral vascular architecture revealed no major differences in microvessel density and perfusion characteristics. However, diameters of intratumoral microvessels were smaller in tumors grown in Ang-2-deficient mice, and the vasculature had an altered pattern of pericyte recruitment and maturation. Ang-2-deficient tumor vessels had higher pericyte coverage indices. Recruited pericytes were desmin and NG2 positive and predominately alpha-smooth muscle actin negative, indicative of a more mature pericyte phenotype. Collectively, the experiments define the role of Ang-2 during tumor angiogenesis and establish a better rationale for combination therapies involving Ang-2 manipulatory therapies.
