Polyphosphate: a fundamental regulator of protein folding in human osteoblasts

Abstract

Polyphosphate (polyP) is structurally simple as a linear chain of phosphate residues linked by ATPlike bonds. Yet, polyP critically affects a range of fundamental biological processes with vague understanding of its mechanistic action in higher eukaryotes. Our study aims to identify significant polyPprotein interactions to investigate polyP’s function and mechanism of action in human osteoblasts. Coextraction of endogenous polyPprotein complexes and pulldown of polyPinteracting proteins via an affinity chromatography approach identified cyclophilins as specific polyPinteracting proteins in SaOS2 osteoblastlike cells. One evident interaction was that of polyP and cyclophilin B (CypB), a chaperone localized in endoplasmic reticulum (ER) and key to collagen folding. Independent biochemical assays show that polyP binds tightly to purified, recombinant CypB and strongly inhibits CypB’s peptidylprolyl cistrans isomerase activity. PolyP caused the assembly of CypB monomers into a multimeric complex which may have masked CypB’s catalytic sites and hindered interactions between CypB with corresponding substrates or cochaperones. We report evidence of polyPCypB colocalization in ER of nondifferentiating SaOS2 cells via confocal microscopy. PolyP’s intracellular effects on CypB’s collagen folding activity in ER of nondifferentiating SaOS2 cells were further investigated using two approaches. Spermine was used as a polyP sequestering agent to hinder endogenous polyPCypB interactions. A vector was constructed to target ERpolyP reduction by transient expression of ERtargeted polyphosphatase. PolyP sequestration and reduction correlates to confirm that endogenous polyP inhibits CypB’s collagen folding activity. This study provides mechanistic insight into how polyP adds a new layer of regulation to protein folding in human cells.