Pathogenesis of Noonan Syndrome is Modulated by NOC2L, a Novel Interactor of LZTR1 Leading to Impaired P53 Signalling.

Abstract

Monoallelic dominant-negative LZTR1 gene variants have been implicated as a cause of Noonan syndrome resulting from hyperactivation of canonical RAS-MAPK signalling. Missense LZTR1 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. Single nucleotide substitutions were generated by mutagenesis of an N-terminal MYC tagged-LZTR1-cDNA. Wild-type and variant constructs were expressed in mammalian cells and lysates prepared for phosphoproteomics. Analysis of transcriptomic data was conducted using Ingenuity-Pathway-Analysis. Significant phospho-peptides, protein-protein interactions, and pathways of interest were probed using immunoblotting, immunofluorescence, nanoluciferase assays and in silico modelling. Two heterozygous LZTR1 variants, affiliated with short stature, were shown in vitro to be thermodynamically stable and associated with elevated pan-Ras levels. Phosphoproteomics revealed upregulation of the histone acetyltransferase inhibitor, NOC2L, in both variants. This finding, consistent on immunoblotting and immunofluorescence, was associated with impaired p53 acetylation. Major effectors of the DNA damage response were preferentially activated in LZTR1 variants. Despite an apparent activation of the DNA damage response and diminished p53 activity, levels of LC3 and phosphorylated-p70 S6 kinase were increased. In silico structure modelling and nanoluciferase assays suggested that LZTR1 interacts with NOC2L, an interaction disrupted in both LZTR1 variants. NOC2L and p53 form a complex that dictates p53 activation. We demonstrate a previously unknown interaction between NOC2L and LZTR1 and hypothesize that LZTR1 acts as a binding factor modulating activity of this complex. Because NOC2L negatively regulates p53, its upregulation leads to p53-mediated transcription inhibition. LZTR1 attenuation resulting from genetic mutations associated with Noonan syndrome, potentiate NOC2L activity leading to reduced apoptosis and a compensatory increase in autophagy.