Deleterious mutations in the X-linked Patched domain-containing 1 (PTCHD1) gene may account for up to 1% of autism cases. Despite this, the PTCHD1 protein remains poorly understood. Structural similarities to Patched family proteins point to a role in sterol transport, but this hypothesis has not been verified experimentally. Additionally, PTCHD1 has been suggested to be involved in Hedgehog signalling, but thus far, the experimental results have been conflicting. To enable a variety of biochemical and structural experiments, we developed a method for expressing PTCHD1 in Spodoptera frugiperda cells, solubilising it in glycol-diosgenin, and purifying it to homogeneity. In vitro and in silico experiments show that PTCHD1 function is not interchangeable with Patched 1 (PTCH1) in canonical Hedgehog signalling, since it does not repress Smoothened in Ptch1−/− mouse embryonic fibroblasts and does not bind Sonic Hedgehog. However, we found that PTCHD1 binds cholesterol similarly to PTCH1. Furthermore, we identified 13 PTCHD1-specific protein interactors through co-immunoprecipitation and demonstrated a link to cell stress responses and RNA stress granule formation. Thus, our results support the notion that despite structural similarities to other Patched family proteins, PTCHD1 may have a distinct cellular function.
The canonical Hedgehog (Hh) signalling pathway is essential for vertebrate development and its uncontrolled activation is a common occurrence in human cancers. Hh signalling converges in the modification of a family of transcription factors, GLI1, GLI2 and GLI3, to orchestrate a cell type and context-specific transcriptional response. Despite binding to very similar responsive elements, the GLI family members can exert diverse and even opposing functions. A recent article by Tolosa et al. (Biochem. J. 477, 3131–3145, 2020) reveals an unexpected layer of complexity, through physical and functional interaction between GLI1 and GLI2. This commentary discusses the biological significance of the findings and incorporates them into an updated ‘GLI code'.
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.
With the importance of Hedgehog signalling in embryonic development, tissue homeostasis and disease, understanding the molecular mechanisms of signal transduction is paramount for the design of specific, effective therapeutics. The Hedgehog receptor PTCH1 and the less studied PTCH2 isoform are evolutionarily related to bacterial RND permeases and sterol sensing proteins, which mobilise hydrophobic compounds powered by a cation gradient. Here we demonstrate that, in the active state, PTCH1 and PTCH2 form homomeric and heteromeric complexes that are inhibited by binding of Sonic Hedgehog. We show that PTCH2, unlike PTCH1, appears to have minimal cholesterol transport activity, but that conserved residues involved in cation transport are essential for its function. Heteromeric PTCH1-PTCH2 complexes depend on PTCH1 cholesterol transport capacity, but the cation transport can be provided in trans by PTCH2, suggesting that some deleterious mutations in either isoform can be silenced by formation of heteromers, enhancing the robustness of this signal transduction system. These findings provide the molecular basis for the intriguing behaviour of PTCH2 as semi-redundant and partially overlapping in function with PTCH1 and explain the dominant negative effect of mutations that disrupt the PTCH2 cation transport triad in rare cases of cancer.
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