Recombinant Mouse Protein SZT2 (Szt2), partial

What is SZT2 and what is its genomic structure?

SZT2 is a gene initially identified in a chemical mutagenesis screen that confers low seizure threshold to mice. The gene spans 72 exons and encodes a large transcript (>10 kb) . The corresponding protein is 378 kD with no significant sequence similarities to any other protein, making it structurally unique . In mammals, the SZT2 gene resides in a highly conserved head-to-head configuration with Med8 (which encodes a Mediator complex subunit), separated by only 91 nucleotides .

What is the expression pattern of SZT2?

SZT2 is transcribed in multiple tissues, with the highest expression observed in the brain . It is also expressed during embryonic development, suggesting roles in both developmental processes and adult neurological function . Northern blot analysis using a 685bp probe containing a portion of the SZT2 transcript from exons 69–72 has been utilized to study its expression in different tissues .

How is SZT2 evolutionarily conserved?

SZT2 demonstrates remarkable evolutionary conservation, with clear, single orthologues found in all land vertebrates and in many invertebrates . This high degree of conservation across species strongly suggests that SZT2 serves a fundamental biological function that has been maintained throughout evolution .

What is the role of SZT2 in cellular signaling?

SZT2 functions as the core subunit of the KICSTOR complex (consisting of KPTN, ITFG2, C12orf66, and SZT2), which negatively regulates mTORC1 signaling . It recruits a fraction of mammalian GATOR1 and GATOR2 to form the SZT2-Orchestrated GATOR (SOG) complex with an essential role in GATOR- and Sestrin-dependent nutrient sensing and mTORC1 regulation . The SOG complex is required for lysosomal localization of these regulatory components .

How does SZT2 interact with other proteins?

SZT2 directly interacts with components of both GATOR1 (DEPDC5, NPRL2, and NPRL3) and GATOR2 (MIOS, WDR24, WDR59) . These interactions are cooperative - SZT2-GATOR2 interaction is lost in the absence of NPRL3, and SZT2-GATOR1 interaction is substantially diminished in the absence of WDR59, WDR24, and MIOS . Size-exclusion chromatography reveals that SZT2 is enriched in fractions with a peak molecular weight around 1.06 MDa, consistent with the formation of a SOG complex .

What happens in the absence of functional SZT2?

SZT2 deficiency results in constitutive mTORC1 signaling even under nutrient deprivation conditions . In mice, SZT2 deficiency leads to neonatal lethality associated with failed mTORC1 inactivation during fasting . Experimentally, Szt2 knockout mice present with spontaneous seizures, indicating its crucial role in maintaining normal neurological function .

How are SZT2 variants associated with epilepsy?

Recessive SZT2 variants are associated with developmental and epileptic encephalopathy 18 (DEE-18) and occasionally neurodevelopmental abnormalities (NDD) without seizures . Recent research has expanded this phenotypic spectrum to include partial epilepsy with favorable outcomes without NDD . The severity of the phenotype correlates with the type of mutation - patients with biallelic null mutations present with more severe symptoms compared to those with biallelic missense variants .

What is the genotype-phenotype correlation observed with SZT2 variants?

A clear genotype-phenotype correlation exists for SZT2 variants. Patients with biallelic null mutations present significantly higher frequency of refractory seizures and earlier onset age of seizure than those with biallelic non-null mutations or with biallelic mutations containing one null variant . Patients with monoallelic or biallelic null mutations typically present with severe developmental and epileptic encephalopathy, while those with biallelic missense variants exhibit mild partial epilepsy with favorable outcomes .

What is the molecular mechanism behind SZT2-associated epilepsy?

Loss-of-function of SZT2 causes overactivation of mTORC1 signaling, which is one of the hallmarks of epilepsy and brain malformations . The SZT2 protein directly interacts with the GATOR1 complex, which consists of DEPDC5, NPRL2, and NPRL3 - genes already associated with heterogeneous epilepsy . This mechanistic link between SZT2 dysfunction, mTORC1 hyperactivation, and seizure generation provides insight into the pathophysiology of SZT2-associated epilepsy .

How can recombinant mouse SZT2 protein be produced?

The complete cDNA of SZT2 can be cloned from mouse cells and subcloned into lentiviral vectors (e.g., pLJM1) with appropriate tags (e.g., FLAG tag) . For protein production, the construct can be transfected into mammalian cells (e.g., HEK293T) or used to generate stable cell lines via lentiviral transduction followed by puromycin selection . Transient transfection systems using vectors like pCMV-HA and pCMV-FLAG can also be employed for expression studies .

What techniques are employed to study SZT2 protein interactions?

Several sophisticated techniques can be used to study SZT2 interactions:

• Co-immunoprecipitation: Using anti-FLAG antibodies to pull down tagged SZT2 and detect interacting partners

• Size-exclusion chromatography (SEC): To separate protein complexes based on molecular weight

• Sucrose density gradient centrifugation: To separate protein complexes based on density

• Crosslinking-assisted immunoprecipitation: To stabilize and detect transient protein interactions

• Mass spectrometry: To identify novel interacting partners in an unbiased manner

How can SZT2 localization be studied in cells?

Due to the low abundance of SZT2 protein, conventional immunofluorescence may be insufficient. Enhanced detection methods such as the Tyramide SuperBoost Kit can be used to amplify the signal . The protocol involves:

• Fixation and permeabilization of cells

• Quenching endogenous HRP activity with 3% hydrogen peroxide

• Blocking with 10% goat serum

• Incubation with anti-FLAG antibody (1:200 dilution)

• Application of poly-HRP-conjugated secondary antibody

• Signal amplification with tyramide working solution

• Counterstaining with organelle markers (e.g., Lamp1 or Lamp2 for lysosomes)

How can SZT2 function be investigated in mouse models?

Researchers investigating SZT2 in mouse models should consider several approaches:

• Gene knockout studies: Homozygous knockout of Szt2 in mice leads to maximal tonic hindlimb extension seizures and preweaning lethality with incomplete penetrance, while heterozygous knockout causes minimal clonic seizures

• Gene-trap mutations: Alternative to complete knockout, gene-trap mutations in Szt2 can also confer low seizure threshold but with different severity than ENU-induced mutations

• Conditional knockouts: For studying tissue-specific effects without lethality

• Knock-in models: To study specific disease-associated variants

What analytical techniques are crucial for SZT2 protein characterization?

Advanced analytical techniques for SZT2 characterization include:

• MALDI-TOF mass spectrometry: For peptide identification of in vitro expressed SZT2-GFP

• Protein purification strategies: Including reduction and alkylation of cysteines, in-gel digestion with trypsin, peptide extraction, and reverse-phase chromatography using C18 zip-tips

• Structural analysis: To understand how mutations might affect protein folding and stability

How can researchers investigate SZT2's role in the mTORC1 pathway therapeutically?

Therapeutic investigations should focus on:

• Rescue experiments: mTORC1 hyperactivation in SZT2-deficient cells can be partially corrected by overexpression of the GATOR1 component DEPDC5, or by lysosome-targeted GATOR2 component WDR59 or lysosome-targeted Sestrin2

• Drug screening: Identifying compounds that can restore normal mTORC1 signaling in SZT2-deficient cells

• Genotype-specific approaches: Developing targeted therapies based on the type of SZT2 mutation (null vs. missense)

• Animal model testing: Evaluating whether mTORC1 inhibitors like rapamycin can ameliorate neurological phenotypes in SZT2-deficient animals