Deamidation enables pathogenic SMAD6 variants to activate the BMP signaling pathway

The BMP signaling pathway plays a crucial role in regulating early embryonic development and tissue homeostasis.SMAD6 encodes a negative regulator of BMP,and rare variants of SMAD6 are recurrently found in individuals with birth defects.However,we observed that a subset of rare pathogenic variants of SMAD6 consistently exhibited positive regulatory effects instead of the initial negative effects on the BMP signaling pathway.We sought to determine whether these SMAD6 variants have common pathogenic mechanisms.Here,we showed that pathogenic SMAD6 variants accompanying this functional reversal exhibit similar increases in deamidation.Mechanistically,increased deamidation of SMAD6 variants promotes the accumulation of the BMP receptor BMPR1A and the formation of new complexes,both of which lead to BMP signaling pathway activation.Specifically,two residues,N262 and N404,in SMAD6 were identified as the crucial sites of deamidation,which was catalyzed primarily by glutamine-fructose-6-phosphate transaminase 2(GFPT2).Additionally,treatment of cells harboring SMAD6 variants with a deamidase inhibitor restored the inhibitory effect of SMAD6 on the BMP signaling pathway.Conversely,when wild-type SMAD6 was manually simulated to mimic the deamidated state,the reversed function of activating BMP signaling was reproduced.Taken together,these findings show that deamidation of SMAD6 plays a crucial role in the functional reversal of BMP signaling activity,which can be induced by a subset of various SMAD6 variants.Our study reveals a common pathogenic mechanism shared by these variants and provides a potential strategy for preventing birth defects through deamidation regulation,which might prevent the off-target effects of gene editing.

Endorepellin和neurexin互作促进神经上皮细胞自噬并维持正常神经管发育

Heparan sulfate proteoglycan 2(HSPG2)gene encodes the matrix protein Perlecan,and genetic inactivation of this gene creates mice that are embryonic lethal with severe neural tube defects(NTDs).We discovered rare genetic variants of HSPG2 in 10%cases compared to only 4%in controls among a cohort of 369 NTDs.Endorepellin,a peptide cleaved from the domain V of Perlecan,is known to promote angiogenesis and autophagy in endothelial cells.The roles of enderepellin in neurodevelopment remain unclear so far.Our study revealed that endorepellin can migrate to the neuroepithelial cells and then be recognized and bind with the neuroepithelia receptor neurexin in vivo.Through the endocytic pathway,the interaction of endorepellin and neurexin physiologically triggers autophagy and appropriately modulates the differentiation of neural stem cells into neurons as a blocker,which is necessary for normal neural tube closure.We created knock-in(KI)mouse models with human-derived HSPG2 variants,using sperm-like stem cells that had been genetically edited by CRISPR/Cas9.We realized that any HSPG2 variants that affected the function of endorepellin were considered pathogenic causal variants for human NTDs given that the severe NTD phenotypes exhibited by these KI embryos occurred in a significantly higher response frequency compared to wildtype embryos.Our study provides a paradigm for effectively confirming pathogenic mutations in other genetic diseases.Furthermore,we demonstrated that using autophagy inhibitors at a cellular level can repress neuronal differentiation.Therefore,autophagy agonists may prevent NTDs resulting from failed autophagy maintenance and neuronal over-differentiation caused by deleterious endorepellin variants.