Inorganic polyphosphate: a mediator of protein folding in osteoblasts via interaction with cyclophilin B

Abstract

Inorganic polyphosphate (polyP), a structurally simple polymer of phosphates linked by high-energy bonds found in ATP, is fundamental in all cells. Far from being molecules with a simple function, evidence is emerging that polyP critically influences a diverse set of key processes in mammalian cells. However, many unanswered questions remain regarding polyP’s mechanism of action in higher eukaryotes and progress in this study is hampered by the lack of tools to analyse polyP function. Using biochemical techniques, we aimed to identify significant polyP-protein interactions to understand polyP’s function in osteoblasts. An affinity chromatography approach, developed by cross-linking polyP to biotin and immobilising polyP via biotin-streptavidin interactions, identified polyP-specific binding proteins in SaOS-2 osteoblast-like cells. We report evidence that cyclophilin B (CypB), a chaperone involved in protein folding and secretion of folded proteins to the extracellular matrix, specifically interacts with polyP at high affinity. In one line of evidence, we used a highly-selective benzimidazolinium dye for long-chain polyP and observed co-localisation of polyP and CypB in the endoplasmic reticulum and secretory vesicles of SaOS-2 cells via confocal microscopy. In another line of evidence, independent binding experiments showed that polyP binds tightly to purified, recombinant human CypB. Biochemical assays using purified CypB further revealed that long-chain polyP strongly inhibits the peptidyl-prolyl cis-trans isomerase activity of CypB. This study provides mechanistic insight into how polyP affects protein folding mediated by CypB function in osteoblasts.