Glycosaminoglycans bind human IL-27 and regulate its activity.

IL-27 is a cytokine of the IL-12 family, composed of EBI3 and IL-27p28. IL-27 regulates immune responses and also other physiological processes including hematopoiesis, angiogenesis, and bone formation. Its receptor, composed of IL-27Rα and gp130, activates the STAT pathway. Here, we show that different glycosaminoglycans (GAGs) modulate human IL-27 activity in vitro. We find that soluble heparin and heparan sulfate efficiently inhibit human IL-27 activity as shown by decreased STAT signaling and downstream biological effects. In contrast, membrane-bound heparan sulfate seems to positively regulate IL-27 activity. Our biochemical studies demonstrate that soluble GAGs directly bind to human IL-27, consistent with in silico analyses, and prevent its binding to IL-27Rα. Although murine IL-27 also bound to GAGs in vitro, its activity was less efficiently inhibited by soluble GAGs. Lastly, we show that two heparin-derivatives, low molecular weight heparin and fondaparinux, that like unfractionated heparin are used in clinics, had weaker or no effect on human IL-27 activity. Together, our data identify GAGs as new players in the regulation of human IL-27 activity that might act under physiological conditions and may also have a clinical impact in heparin-treated patients.

A proximity-labeling proteomic approach to investigate invadopodia molecular landscape in breast cancer cells.

Metastatic progression is the leading cause of mortality in breast cancer. Invasive tumor cells develop invadopodia to travel through basement membranes and the interstitial matrix. Substantial efforts have been made to characterize invadopodia molecular composition. However, their full molecular identity is still missing due to the difficulty in isolating them. To fill this gap, we developed a non-hypothesis driven proteomic approach based on the BioID proximity biotinylation technology, using the invadopodia-specific protein Tks5α fused to the promiscuous biotin ligase BirA* as bait. In invasive breast cancer cells, Tks5α fusion concentrated to invadopodia and selectively biotinylated invadopodia components, in contrast to a fusion which lacked the membrane-targeting PX domain (Tks5ß). Biotinylated proteins were isolated by affinity capture and identified by mass spectrometry. We identified known invadopodia components, revealing the pertinence of our strategy. Furthermore, we observed that Tks5 newly identified close neighbors belonged to a biologically relevant network centered on actin cytoskeleton organization. Analysis of Tks5ß interactome demonstrated that some partners bound Tks5 before its recruitment to invadopodia. Thus, the present strategy allowed us to identify novel Tks5 partners that were not identified by traditional approaches and could help get a more comprehensive picture of invadopodia molecular landscape.