The pathogenesis of Noonan syndrome is modulated by NOC2L, a novel interactor of LZTR1 leading to impaired p53 signalling

This study identifies a new interaction between NOC2L and LZTR1 that may affect p53 signaling in Noonan syndrome, suggesting a complex pathway that warrants further investigation.

Summary and findings

This study investigates the role of LZTR1 variants in Noonan syndrome (NS) and their impact on p53 signaling. Two heterozygous LZTR1 variants were expressed in mammalian cells, revealing interactions with NOC2L and effects on p53 acetylation. The findings suggest a complex relationship between these proteins that may influence NS pathogenesis.

Abstract

<title>Abstract</title> <p><bold>Introduction</bold>: Monoallelic dominant negative <italic>LZTR1</italic> gene variants have been implicated as a cause of NS due to hyperactivation of the canonical RAS-MAPK signalling pathway. Missense <italic>LZTR1 </italic>variants have been associated with defective ubiquitination theoretically leading to increased Ras substrate availability and altered p53 signalling. We investigated the role of LZTR1 in this pathway. <bold>Methods</bold>: Single nucleotide substitutions were created by mutagenesis of an N-terminal MYC tagged-<italic>LZTR1</italic> cDNA. WT and variant constructs were expressed in mammalian cells and lysates prepared for phosphoproteomic analysis and immunoblotting. Analysis of transcriptomic data was conducted using Ingenuity Pathway Analysis. Significant phospho-peptides, protein-protein interactions and pathways of interest were probed using western blotting, immunofluorescence, nanoluciferase assays and <italic>in silico</italic> prediction tools. <bold>Results</bold>: Two heterozygous <italic>LZTR1</italic> variants, which segregated with short stature and features of growth hormone insensitivity (p.K156E, p.G248R), were expressed in a mammalian cell line. Both variants were thermodynamically stable and associated with elevated cytoplasmic levels of pan-Ras. Phosphoproteomic assays revealed upregulation of the histone acetyltransferase inhibitor, NOC2L (NOC2 Like Nucleolar Associated Transcriptional Repressor), in both variants. This finding, consistent upon immunoblotting and immunofluorescence, was associated with impaired acetylation of p53, with reduced levels of acetylated lysine residue 382 in both mutants. Furthermore, Ataxia Telangiectasia Mutated (ATM) kinase and Checkpoint kinase 1 (CHK1), major effectors of the DNA damage response (DDR), were preferentially activated in <italic>LZTR1</italic> variants. Despite an apparent activation of the DDR and diminished p53 activity, levels of LC3 and phosphorylated p70 S6 kinase were increased. <italic>In silico</italic> structure modelling suggested that LZTR1 interacts with NOC2L via the central part of the protein and this interaction was validated by nanoluciferase assays and disrupted in both <italic>LZTR1</italic> variants. <bold>Conclusion</bold>: NOC2L and p53 form a complex which dictates p53 activation. We demonstrate a previously unknown interaction between NOC2L and LZTR1 and hypothesise that LZTR1 acts as a binding factor modulating the activity of this complex. As NOC2L negatively regulates p53, upregulation of this protein would lead to p53-mediated transcription inhibition. LZTR1 attenuation due to genetic mutations associated with NS, potentiate NOC2L activity leading to reduced apoptosis and a compensatory increase in autophagy. Given its potential role in the multisystem pathogenesis of NS, NOC2L may represent a novel therapeutic target however, additional work is needed to further characterise its organ-specific effects.</p>

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