Evidence is emerging that inorganic polyphosphate (polyP), a ubiquitous polymer of orthophosphates linked by ATP-like bonds, is fundamental to numerous key biological processes in mammalian cells. PolyP’s involvement in higher eukaryotes includes chaperoning ability and as modifier of protein misfolding events but several unanswered questions remain regarding polyP’s mechanism of action. We aim to identify significant polyP-protein interactions to understand polyP function in osteoblasts. PolyP-specific binding proteins in SaOS-2 osteoblast-like cells were identified using an affinity chromatography approach developed by crosslinking polyP-biotin and immobilising polyP via biotin-streptavidin interactions. We report evidence that cyclophilin B (CypB), an endoplasmic reticulum (ER)-localised chaperone and key enzyme in collagen folding, functionally interacts with polyP at high affinity. Independent binding assays validated that polyP binds tightly to purified, recombinant human CypB. Rotamase assays further revealed that polyP strongly inhibits CypB’s peptidyl-prolyl cis-trans isomerase activity. Confocal microscopy confirmed polyP-CypB colocalisation in the ER of SaOS-2 cells. To further study polyP’s intracellular activity, we investigated polyP’s effects on CypB’s collagen folding activity in non-differentiating SaOS-2 cells. Spermine was used to sequester polyP and obstruct polyP-CypB binding. A vector was constructed to express ER-targeted polyphosphatase resulting in reduction of ER-polyP levels. Results suggest that CypB-mediated collagen folding was enhanced by polyP sequestration and depletion in ER. This study provides mechanistic insight into how polyP affects protein folding mediated by CypB function in osteoblasts.

Poster Presentation: no. P32