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Proteomics analysis unravels new functions of the KICSTOR component SZT2

ABSTRACT: Developmental and epileptic encephalopathy is a group of rare, severe neurodevelopmental disorders characterized by the co-occurrence of epilepsy and intellectual disability (ID), in which there is additional developmental impairment independent of epileptic activity. In 2009, through a chemical mutagenesis screen performed in mice, it was found that mutations in Seizure threshold 2 (SZT2) trigger a higher susceptibility to seizures in a semidominant manner. Since then 27 patients have been identified with biallelic SZT2 mutations (in compound heterozygous or homozygous state), all of them displaying developmental delay of varying severity and dysmorphic features such as macrocephaly. Patients commonly suffer from recurrent epileptic seizures that often are refractory to available therapies. Human SZT2 encodes a 3,375 amino acids protein highly conserved throughout evolution, that is predominantly expressed in embryonal stages and in the adult the brain. The molecular functions of SZT2 protein are largely unknown however it has been reported that SZT2 forms a complex with KPTN, ITFG2, and C12orf66. This complex, named KICSTOR, senses aminoacid depletion and functions as a negative regulator of mTORC1 under catabolic conditions. Taking into consideration that SZT2 remain one of the least characterized regulators of mTORC1 we decided to decipher its interactome under catabolic and anabolic conditions. We found mTORC1 and AMPK signaling components enriched in the SZT2 interactome, but also prominent clusters of autophagy and ciliogenesis regulators as well as a significant number of hits that regulate neurogenesis and neurodegenerative processes. We have validated a few key hits from the interactome, including proteins involved in cilliogenesis and epilepsy. The detailed analysis of the SZT2KOs demonstrated that although these cells show increased mTORC1 signaling that can be prevented by treatment with either rapamycin or torin, they also contra intuitively display increased autophagy that is independent of the physiological conditions tested. These results are in accordance with our interactome data were we detect a significant pool of selective autophagy receptors/regulators. Overall the presented data should serve as a tool to address the physiological functions of SZT2 in more comprehensive manner. In the manuscript discussion we put forward our model in which SZT2 plays a broader role in the coordination of cellular metabolism than previously anticipated.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Kidney Cell

SUBMITTER: Mariana Eca Guimaraes de Araujo

LAB HEAD: Mariana Eca Guimaraes de Araujo

PROVIDER: PXD027662 | Pride | 2021-11-02

REPOSITORIES: Pride

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Project description:Missense mutations in the TP53 gene are frequent genetic alterations in human tumor tissue and cell lines. In contrast to wild-type p53, the mutant p53 (mutp53) protein has lost the transcriptional activity towards pro-apoptotic and growth arrest genes, but retained the property to interact with DNA in a structure-specific fashion. Expression of mutp53 is advantageous for tumor cells, however the molecular mechanism of mutp53 action is still not known. We used the glioblastoma-derived U-251 MG human cell line to analyze DNA binding of mutant p53 (R273H mutation) on a Nimblegen custom 135k tiling array and to correlate mutp53 binding regions with the epigenetic state and occupation by other transcription factors (ETS1 and SP1). We found that mutp53-binding regions are G/C-rich and are located around transcriptional start sites (TSS) of many protein-coding genes, which in most cases are active, but are not always regulated upon transient mutp53 depletion. We propose a model which does not only rely on interactions of mutp53 with diverse transcriptional regulators at active promoters, but primarily is based on a DNA binding activity of mutp53. We designed a Nimblegen custom 135k tiling array that covers a large set of putative and known p53 (wild-type and mutant) target genes in the human genome. For analysis of the epigenetic state of genes covered by the tiling array in control and mutant p53-depleted U251 cells we focused on changes in active histone marks, H3K4me3 and H3K9Ac, and RNA polymerase II recruitment and processivity. H3K4me3 and H3K9Ac marks are enriched in active promoter regions and the phosphorylation of serine 5 (S5-P) and serine 2 (S2-P) in the CTD of RNA polymerase II have been described to define initiated and elongating complexes, respectively. We performed the ChIP-chip experiments for H3K4me3, H3K9Ac, RNA polymerase II (S5-P) and RNA polymerase II (S2-P) from U251 cells transfected with p53-specific siRNA or control siRNA (2 biological replicates each). To analyze binding of mutant p53 to the genes covered by the tiling array we performed mutant p53 ChIP-chip experiments in 4 biological replicates of. In addition, we analyzed the distribution of SP1 and ETS1 binding sites in 3 biological replicates.

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Proteomics analysis unravels new functions of the KICSTOR component SZT2

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