Integral Membrane Protein 2A Is a Negative Regulator of Canonical and Non-Canonical Hedgehog Signalling

Abstract

The Hedgehog (Hh) receptor PTCH1 and the integral membrane protein 2A (ITM2A) inhibit autophagy by reducing autolysosome formation. In this study, we demonstrate that ITM2A physically interacts with PTCH1; however, the two proteins inhibit autophagic flux independently, since silencing of ITM2A did not prevent the accumulation of LC3BII and p62 in PTCH1-overexpressing cells, suggesting that they provide alternative modes to limit autophagy. Knockdown of ITM2A potentiated PTCH1-induced autophagic flux blockade and increased PTCH1 expression, while ITM2A overexpression reduced PTCH1 protein levels, indicating that it is a negative regulator of PTCH1 non-canonical signalling. Our study also revealed that endogenous ITM2A is necessary for timely induction of myogenic differentiation markers in C2C12 cells since partial knockdown delays the timing of differentiation. We also found that basal autophagic flux decreases during myogenic differentiation at the same time that ITM2A expression increases. Given that canonical Hh signalling prevents myogenic differentiation, we investigated the effect of ITM2A on canonical Hh signalling using GLI-luciferase assays. Our findings demonstrate that ITM2A is a strong negative regulator of GLI transcriptional activity and of GLI1 stability. In summary, ITM2A negatively regulates canonical and non-canonical Hh signalling.

Exploring ITM2A as a new potential target for brain delivery

“…We reasoned that engineering the protein with GFP and HA tags at the C-Ter (extracellular domain) position of the protein, could serve the double purpose of visualizing its cellular localization using GFP fluorescence and allow the study of binding and uptake with antibodies against these tags. This strategy of tagging a protein to circumvent the absence of monoclonal antibodies has actually been reported for ITM2A and has helped decipher its role in the hedgehog signalling pathway [ 40 ]. Tags at the N-Ter were also engineered as controls along with several positions within the extracellular Brichos domain, toward bringing information on the precise site involved in endocytosis.…”

“…Two main workflows have been described for the search of new mechanisms of brain delivery. The first strategy relies on transcriptomic and proteomic approaches from either brain microvessels or endothelial cells of human [ 40 ], cynomolgus monkey [ 41 ], bovine [ 42 ], rat [ 33 , 43 ] or mouse [ 14 , 44 – 47 ], including human [ 34 , 48 – 50 ] diseased brains. A second approach is based on phenotypic in vitro or in vivo screening of antibodies and peptide libraries displayed in various formats including phage and yeast [ 51 – 53 ].…”