J Clin Endocrinol Metab. 2025 Nov 1:dgaf602. doi: 10.1210/clinem/dgaf602. Online ahead of print.
ABSTRACT
INTRODUCTION: Monoallelic dominant-negative LZTR1 gene variants have been implicated as a cause of NS due to 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.
METHODS: Single nucleotide substitutions were generated by mutagenesis of an N-terminal MYC tagged-LZTR1-cDNA. WT 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.
RESULTS: 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 upon immunoblotting and immunofluorescence, was associated with impaired p53 acetylation. Major effectors of the DNA damage response (DDR), were preferentially activated in LZTR1 variants. Despite an apparent activation of the DDR 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.
CONCLUSION: 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 activity of this complex. Since NOC2L negatively regulates p53, its upregulation leads 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.
PMID:41175093 | DOI:10.1210/clinem/dgaf602
