The SZT2 Interactome Unravels New Functions of the KICSTOR Complex

Abstract

Seizure threshold 2 (SZT2) is a component of the KICSTOR complex which, under catabolic conditions, functions as a negative regulator in the amino acid-sensing branch of mTORC1. Mutations in this gene cause a severe neurodevelopmental and epileptic encephalopathy whose main symptoms include epilepsy, intellectual disability, and macrocephaly. As SZT2 remains one of the least characterized regulators of mTORC1, in this work we performed a systematic interactome analysis under catabolic and anabolic conditions. Besides numerous mTORC1 and AMPK signaling components, we identified clusters of proteins related to autophagy, ciliogenesis regulation, neurogenesis, and neurodegenerative processes. Moreover, analysis of SZT2 ablated cells revealed increased mTORC1 signaling activation that could be reversed by Rapamycin or Torin treatments. Strikingly, SZT2 KO cells also exhibited higher levels of autophagic components, independent of the physiological conditions tested. These results are consistent with our interactome data, in which we detected an enriched pool of selective autophagy receptors/regulators. Moreover, preliminary analyses indicated that SZT2 alters ciliogenesis. Overall, the data presented form the basis to comprehensively investigate the physiological functions of SZT2 that could explain major molecular events in the pathophysiology of developmental and epileptic encephalopathy in patients with SZT2 mutations.

Fine-Tuning Cardiac Insulin-Like Growth Factor 1 Receptor Signaling to Promote Health and Longevity

“…The TFEB (transcription factor EB) is another mTORC1 substrate that is intimately linked to lysosome biogenesis and autophagy, 25 but has different phosphorylation kinetics from other mTORC1 substrates. 26 TFEB phosphorylation by mTORC1 reduces the electrophoretic mobility of TFEB, 27 , 28 resulting in an upward shift in the immunoblot signal ( Figure S13A ). dnPI3K mice exhibited a higher TFEB signal at the lower, unphosphorylated molecular weight ( Figure S13A ), suggesting reduced phosphorylation by mTORC1.…”

A CRISPR-screen in intestinal epithelial cells identifies novel factors for polarity and apical transport

“…Total cell lysates were prepared and westernblot analysis was performed as described previously (Cattelani et al, 2021). Cells were washed in 1x cold PBS, scraped from respective culture-plates and pelleted with 1500xg for 5 min at 4°C.…”

“…Total cell lysates were prepared and westernblot analysis was performed as described previously (Cattelani et al, 2021) (fraction V), 1 mM EDTA, 0.05% Tween20, and 0.02% NaN3, and probed with the respective antibodies.…”

Induced Pluripotent Stem Cell (iPSC) Lines from a Family with Resistant Epileptic Encephalopathy Caused by Compound Heterozygous Mutations in SZT2 Gene

“…When SZT2 is deficient, KICSTOR does not assemble, and mTORC1 becomes constitutively localized to the lysosomes and persistently activated, thus promoting biogenesis in the cell even during nutrient shortage [ 3 , 4 ]. Moreover, our recent interactome analysis highlighted the possibility of novel functional roles of SZT2 in additional cellular processes that may or may not be directly mediated by mTORC1, including ciliogenesis, endocytosis, mitochondrial function and neurological disorders [ 5 ].…”