Systematic discovery of protein interaction interfaces using AlphaFold and experimental validation.

Structural resolution of protein interactions enables mechanistic and functional studies as well as interpretation of disease variants. However, structural data is still missing for most protein interactions because we lack computational and experimental tools at scale. This is particularly true for interactions mediated by short linear motifs occurring in disordered regions of proteins. We find that AlphaFold-Multimer predicts with high sensitivity but limited specificity structures of domain-motif interactions when using small protein fragments as input. Sensitivity decreased substantially when using long protein fragments or full length proteins. We delineated a protein fragmentation strategy particularly suited for the prediction of domain-motif interfaces and applied it to interactions between human proteins associated with neurodevelopmental disorders. This enabled the prediction of highly confident and likely disease-related novel interfaces, which we further experimentally corroborated for FBXO23-STX1B, STX1B-VAMP2, ESRRG-PSMC5, PEX3-PEX19, PEX3-PEX16, and SNRPB-GIGYF1 providing novel molecular insights for diverse biological processes. Our work highlights exciting perspectives, but also reveals clear limitations and the need for future developments to maximize the power of Alphafold-Multimer for interface predictions.

Identification of Novel and Potent Modulators Involved in Neonatal Cardiac Regeneration.

Neonatal mammalian heart has been shown to possess the capacity to regenerate substantially after an injury. This remarkable regenerative capacity is lost in a week. This transition has been marked with cardiomyocyte cell cycle arrest and induction of fibrotic response similar to what occurs after myocardial infarction in adult hearts. Recent studies outlined the function of several cardiogenic factors that play a pivotal role in neonatal cardiac regeneration. However, underlying molecular mechanisms of neonatal cardiac regeneration and other cardiogenic factors remained elusive. Here, we investigated the involvement of novel putative cardiogenic factors in neonatal cardiac regeneration and cardiomyocyte cell cycle withdrawal. We have shown that Cbl, Dnmt3a, and Itch are significantly downregulated during neonatal cardiac regeneration process after cardiac injury in vivo. Intriguingly, several of studied factors are upregulated in non-regenerative period of 7-day-old mice after cardiac injury. Knockdown of Cbl, Dnmt3a and Itch in rat neonatal cardiomyocytes lead to the induction of cardiomyocyte proliferation. Cardiomyocyte proliferation accompanies upregulation of positive regulators of cardiomyocyte division and downregulation of CDKIs. Taken together, our findings suggest that Cbl, Dnmt3a, and Itch may be involved in the regulation of cardiomyocyte cell cycle withdrawal and may represent new targets for the induction of cardiac regeneration.