AUTHOR=Hoffmann Felix , Bolz Sylvia , Junger Katrin , Klose Franziska , Stehle Isabel F. , Ueffing Marius , Boldt Karsten , Beyer Tina 

TITLE=Paralog-specific TTC30 regulation of Sonic hedgehog signaling

JOURNAL=Frontiers in Molecular Biosciences

VOLUME=Volume 10 - 2023

YEAR=2023

URL=https://www.frontiersin.org/journals/molecular-biosciences/articles/10.3389/fmolb.2023.1268722

DOI=10.3389/fmolb.2023.1268722

ISSN=2296-889X

ABSTRACT=The intraflagellar transport machinery is essential for cilia assembly, maintenance and importantly also for trans-localization of signaling proteins. The IFT machinery consists of two large multiprotein complexes, one of which is the IFT-B. TTC30A and TTC30B are integral components of this complex and were shown before to have redundant function in context of IFT, preventing the disruption of IFT-B and thus a severe ciliogenesis defect upon loss of one paralogue. In this study, we re-analyzed the paralogue specific protein complexes and discovered a potential involvement of TTC30A or TTC30B in ciliary signaling. Specifically, we investigated a TTC30A specific interaction with protein kinase A catalytic subunit α, a negative regulator of Sonic hedgehog signaling (Shh). Defects in this ciliary signaling pathway are often correlated to synpolydactyly, which intriguingly is also linked to a rare TTC30 variant. For an in-depth analysis of this unique interaction and the influence on Shh, TTC30A or B single- and double-knockout hTERT-RPE1 were employed as well as rescue cells harboring wildtype TTC30 or the corresponding mutation. We could show that mutant TTC30A inhibits ciliary localization of Smoothened. This observed effect is independent of Patched1 but associated with a distinct phosphorylated PKA substrate accumulation upon treatment with Forskolin. This rather prominent phenotype was attenuated in mutant TTC30B. Mass spectrometry analysis of wildtype versus mutated TTC30A or TTC30B uncovered differences in protein complex pattern with identifying an impaired TTC30A-IFT57 interaction as possible link leading to synpolydactyly. We could observe no impact on cilia assembly leading to the hypothesis, that a slight decrease in IFT-B binding can be compensated but mild phenotypes, like synpolydactyly, can be induced by subtle signaling changes. Our systematic approach revealed the paralogue specific influence of TTC30A KO and mutated TTC30A on the activity of PRKACA as well as the uptake of Smoothened into the cilium resulting in a downregulation of Shh. This downregulation combined with interactome alterations suggest a potential mechanism of how mutant TTC30A is linked to synpolydactyly.