ALDH1A (aldehyde dehydrogenase 1A), agonist and catalysate of ALDH1A, application of inhibitor
A technology for catalytic products and agonists, which can be used in the screening of compounds, active ingredients of heterocyclic compounds, biochemical equipment and methods, etc., and can solve problems that cannot be used to explain the mechanism of autism.
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[0147] 18. Virus Preparation and Brain-Targeted Injection
[0148] All indicated genes are driven by the Synapsin I (SynI) promoter and have a Flag tag fused to the N-terminus. The envelope protein serotype of adeno-associated virus (AAV) is AAV2 / 9, which is packaged by Shanghai Heyuan Biotechnology Company (Obio). AAV-SynI-Flag-UBE3A and AAV-SynI-Flag-UBE3A-T508E virus titers were approximately 1.5×10 13 Copy number / mL, AAV-SynI-Flag-EGFP virus titer is about 9.5×10 12 Copy number / mL.
[0149] 3-week-old mice were anesthetized by intraperitoneal injection of pentobarbital (50 mg / kg), and then fixed on a locator (Rivold, China). AAV virus diluted in 1 μL PBS was injected with a quantitative syringe pump (Stoelting) at a speed of 0.2 μL / min on both sides of the PFC area in the inner side of the mouse brain. Positions of targeted injections (relative to the bregma): AP, +2 mm; ML, ±0.5 mm; DV, -1.3 mm. The amount of virus injected at each injection point was: AAV-SynI-Flag-...
Embodiment 1
[0166] Example 1 The copy number amplification of chromosome 15q11.2-14 was found in the samples of autism patients
[0167] Three clinically diagnosed patients with autism phenotype were collected in the early stage, and the amplification of chromosome 15q11.2-14 ( figure 1 a-b). A series of genes are included in this expanded region, and their association with autism is not clear, except for the UBE3A gene which is generally confirmed to play a key role in the 15q expansion syndrome ( figure 1 b-c). The peripheral blood lymphocytes of these patients were established into immortalized cell lines by EBV virus infection method, so as to facilitate the follow-up functional research. Among them, compared with the control cells from healthy volunteers, the expression levels of UBE3A protein in these three autistic cells were significantly increased ( figure 1 d).
Embodiment 2
[0168] Example 2 Screening out the new substrate ALDH1A protein family of UBE3A
[0169] Since there is no known substrate of UBE3A that can explain the association between the pathogenesis of ASD and the overactivation of UBE3A from a mechanistic point of view (Glessner et al., 2009; Sell et al., 2015), the human UBE3A protein was first used as a bait, Unknown substrate proteins were screened by yeast two-hybrid method. Among them, many positive clones were found to carry the cDNA encoding the rate-limiting enzyme ALDH1A2 for RA synthesis ( figure 2 a). Retinoic acid RA, as an active metabolite of vitamin A (retinol), is required for the development and growth of higher animals including humans (Shearer et al., 2012). The synthesis of RA is completed by two steps, including retinol dehydrogenase (RDH10) that catalyzes retinol to retinal, and retinal dehydrogenase (ALDH1A1,2) that catalyzes retinal to retinoic acid. , 3 families). Co-immunoprecipitation experiments verifi...
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