Dataset Information

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.

SUBMITTER: Cattelani C

PROVIDER: S-EPMC8534408 | biostudies-literature | 2021 Oct

REPOSITORIES: biostudies-literature

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The SZT2 Interactome Unravels New Functions of the KICSTOR Complex.

Cattelani Cecilia C Lesiak Dominik D Liebscher Gudrun G Singer Isabel I II Stasyk Taras T Wallnöfer Moritz H MH Heberle Alexander M AM Corti Corrado C Hess Michael W MW Pfaller Kristian K Kwiatkowski Marcel M Pramstaller Peter P PP Hicks Andrew A AA Thedieck Kathrin K Müller Thomas T Huber Lukas A LA Eca Guimaraes de Araujo Mariana M

Cells 20211009 10

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 anabol ...[more]

PMID: 34685691

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Project description: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.

Project description:BackgroundEmergence of drug resistant varieties of tuberculosis is posing a major threat to global tuberculosis eradication programmes. Although several approaches have been explored to counter resistance, there has been limited success due to a lack of understanding of how resistance emerges in bacteria upon drug treatment. A systems level analysis of the proteins involved is essential to gain insights into the routes required for emergence of drug resistance.ResultsWe derive a genome-scale protein-protein interaction network for Mycobacterium tuberculosis H37Rv from the STRING database, with proteins as nodes and interactions as edges. A set of proteins involved in both intrinsic and extrinsic drug resistance mechanisms are identified from literature. We then compute shortest paths from different drug targets to the set of resistance proteins in the protein-protein interactome, to derive a sub-network relevant to study emergence of drug resistance. The shortest paths are then scored and ranked based on a new scheme that considers (a) drug-induced gene upregulation data, from microarray experiments reported in literature, for the individual nodes and (b) edge-hubness, a network parameter which signifies centrality of a given edge in the network. High-scoring paths identified from this analysis indicate most plausible pathways for the emergence of drug resistance. Different targets appear to have different propensities for four drug resistance mechanisms. A new concept of 'co-targets' has been proposed to counter drug resistance, co-targets being defined as protein(s) that need to be simultaneously inhibited along with the intended target(s), to check emergence of resistance to a given drug.ConclusionThe study leads to the identification of possible pathways for drug resistance, providing novel insights into the problem of resistance. Knowledge of important proteins in such pathways enables identification of appropriate 'co-targets', best examples being RecA, Rv0823c, Rv0892 and DnaE1, for drugs targeting the mycolic acid pathway. Insights obtained about the propensity of a drug to trigger resistance will be useful both for more careful identification of drug targets as well as to identify target-co-target pairs, both implementable in early stages of drug discovery itself. This approach is also inherently generic, likely to significantly impact drug discovery.

Project description:UnlabelledMycobacteriophages are viruses that infect mycobacterial hosts and are prevalent in the environment. Nearly 700 mycobacteriophage genomes have been completely sequenced, revealing considerable diversity and genetic novelty. Here, we have determined the protein complement of mycobacteriophage Giles by mass spectrometry and mapped its genome-wide protein interactome to help elucidate the roles of its 77 predicted proteins, 50% of which have no known function. About 22,000 individual yeast two-hybrid (Y2H) tests with four different Y2H vectors, followed by filtering and retest screens, resulted in 324 reproducible protein-protein interactions, including 171 (136 nonredundant) high-confidence interactions. The complete set of high-confidence interactions among Giles proteins reveals new mechanistic details and predicts functions for unknown proteins. The Giles interactome is the first for any mycobacteriophage and one of just five known phage interactomes so far. Our results will help in understanding mycobacteriophage biology and aid in development of new genetic and therapeutic tools to understand Mycobacterium tuberculosis.ImportanceMycobacterium tuberculosis causes over 9 million new cases of tuberculosis each year. Mycobacteriophages, viruses of mycobacterial hosts, hold considerable potential to understand phage diversity, evolution, and mycobacterial biology, aiding in the development of therapeutic tools to control mycobacterial infections. The mycobacteriophage Giles protein-protein interaction network allows us to predict functions for unknown proteins and shed light on major biological processes in phage biology. For example, Giles gp76, a protein of unknown function, is found to associate with phage packaging and maturation. The functions of mycobacteriophage-derived proteins may suggest novel therapeutic approaches for tuberculosis. Our ORFeome clone set of Giles proteins and the interactome data will be useful resources for phage interactomics.

Dataset Information

The SZT2 Interactome Unravels New Functions of the KICSTOR Complex.

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The SZT2 Interactome Unravels New Functions of the KICSTOR Complex.

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