PMID:24466266

Citation	Hunter, MC, O'Hagan, KL, Kenyon, A, Dhanani, KC, Prinsloo, E and Edkins, AL (2014) Hsp90 Binds Directly to Fibronectin (FN) and Inhibition Reduces the Extracellular Fibronectin Matrix in Breast Cancer Cells. PLoS ONE 9:e86842
Abstract	Heat shock protein 90 (Hsp90) has been identified in the extracellular space and has been shown to chaperone a finite number of extracellular proteins involved in cell migration and invasion. We used chemical cross-linking and immunoprecipitation followed by tandem mass spectrometry (MS/MS) to isolate a complex containing Hsp90 and the matrix protein fibronectin (FN) from breast cancer cells. Further analysis showed direct binding of Hsp90 to FN using an in vitro co-immunoprecipitation assay, a solid phase binding assay and surface plasmon resonance (SPR) spectroscopy. Confocal microscopy showed regions of co-localisation of Hsp90 and FN in breast cancer cell lines. Exogenous Hsp90β was shown to increase the formation of extracellular FN matrix in the Hs578T cell line, whilst knockdown or inhibition of Hsp90 led to a reduction in the levels of both soluble and insoluble FN and could be partially rescued by addition of exogenous Hsp90β. Treatment of cells with novobiocin led to internalization of FN into vesicles that were positive for the presence of the lysosomal marker, LAMP-1. Taken together, the direct interaction between FN and Hsp90, as well as the decreased levels of both soluble and insoluble FN upon Hsp90 inhibition or knockdown, suggested that FN may be a new client protein for Hsp90 and that Hsp90 was involved in FN matrix assembly and/or stability. The identification of FN as a putative client protein of Hsp90 suggests a role for Hsp90 in FN matrix stability, which is important for a number of fundamental cellular processes including embryogenesis, wound healing, cell migration and metastasis.
Links	PubMed Online version:10.1371/journal.pone.0086842
Keywords
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
HUMAN:HS90B	involved_in	GO:0097435: supramolecular fiber organization	ECO:0000315: mutant phenotype evidence used in manual assertion		P		Seeded From UniProt	complete
HUMAN:HS90B		GO:0043206: extracellular fibril organization	ECO:0000315:		P		Fig 7	complete
HUMAN:HS90B		GO:0097435: fibril organization	ECO:0000315:		P		An Interaction observed in vitro and colocalization of Hsp90 and FN in both normal and cancer cell lines were investigated using confocal microscopy and the DOC assay. These methods helped to determine the effect of exogenous Hsp90β on FN matrix formation and morphology in Hs578T cells. Treatment of the Hs578T cell line with exogenous soluble Hsp90β led to an increase in insoluble FN relative to soluble FN, suggesting a role for extracellular Hsp90 in fibril formation. Other methods used include co-immunoprecipitation, solid phase binding assay, immunoblot analysis, SPR spectroscopy and mass spectrometry.	complete CACAO 9330

References

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