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A kind of cortical actin mutant 8KQ and its application

A technology of actin and mutants, applied in the field of protein peptides, can solve the problems of limiting ASD pathogenesis research and clinical drug development, and achieve the effect of improving repetitive stereotyped behavior, fast onset and obvious effect

Active Publication Date: 2019-08-09
NORTHEAST NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Genetic variation presents a high degree of heterogeneity, limiting ASD pathogenesis research and clinical drug development

Method used

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  • A kind of cortical actin mutant 8KQ and its application
  • A kind of cortical actin mutant 8KQ and its application
  • A kind of cortical actin mutant 8KQ and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Excitatory synapses express acetylated cortical actin

[0038] Coat culture dishes or glass slides with polylysine (PLL) in advance, overnight at room temperature or 2 hours at 37°C. The PLL was recovered, rinsed with sterile water for 3 times, and the coated culture dish or glass slide could be stored at 4°C for 1 week. Pre-cool the HBSS buffer, and pay attention to maintain low temperature and sterility before digestion. On the day of birth, the suckling mice were dizzy with ice, sterilized with 75% alcohol, and the brains were taken out quickly. The hippocampal / cortical tissue was stripped in HBSS and gently transferred to EP tubes filled with fresh HBSS. Cut into small pieces, digest with trypsin (0.125%) at 37°C for 30 minutes, and gently resuspend the tissue pieces every 10 minutes during this period. The digestion was terminated with F12 medium containing 10% FBS, centrifuged at 800 rpm for 1 min, and the supernatant was removed. Add F12+10% FBS medium to res...

Embodiment 2

[0042] Studies have shown that the acetylation of cortical actin is regulated by acetyltransferase (PCAF) and sirtuin (HDAC6), and there may be non-enzymatic acetylation. Acetyl coenzyme A (Ac-CoA) is the only source of acetyl groups required for protein acetylation, and the intracellular concentration of Ac-CoA affects the level of acetylation modification of proteins. To explore this possibility and its biological significance, in vitro acetylation experiments were performed. Reaction buffer: Tris-HCl (50 mM, pH 8.0), EDTA (0.1 mM), DTT (1 mM), glycerol (10%), sodium butyrate (10 mM), add protease inhibitors before use. Reaction substrate: cortical actin, acetyl donor Ac-CoA. The reaction system was incubated at 30°C for 1 hour, and the reaction product obtained was used for Western blot antibody detection. The antibody used was the pan-acetylated antibody acetylated-lysine; the added amount of the substrate cortical actin was indicated by Coomassie brilliant blue staining....

Embodiment 3

[0044] Identification of acetylation sites in cortical actin

[0045] In order to further explore the role of cortical actin acetylation in the regulation of postsynaptic structure and function, LC-MS / MS method was used to identify the in vitro acetylation assay ( figure 2 ) Acetylation site of actin in the middle cortex. The SDS strip at the location of the cortical actin shown by Coomassie brilliant blue staining was excised, and the subsequent LC-MS / MS process was completed by Hangzhou Jingjie Biotechnology Co., Ltd. The specific process is as follows: the glue strips were chopped, and the glue pieces were decolorized with 50% acetonitrile containing 50 mM ammonium bicarbonate (NH4HCO3). The gel pieces were dehydrated by incubating with 100% acetonitrile for 5 min, and then the liquid phase in the system was removed, a dithiothreitol solution with a final concentration of 10 mM was added, and incubated at 37° C. for 60 min. Incubate again with 100% acetonitrile for dehyd...

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Abstract

The invention provides a cortex actin mutant 8KQ and an application, and belongs to the technical field of protein polypeptide. The cortex actin mutant 8KQ has an amino acid sequence as shown in SEQIDNo:1. The cortex actin mutant 8KQ lies in that 8 lysine sites of K107, K152, K171, K181, K193, K235, K309 and K314 of cortex actin (NP_031829.2) are mutated into glutamine, and the charge characteristics of protein under the acetylation state are simulated. The cortex actin mutant 8KQ can be successfully expressed in cultured cells, can reduce combining capacity with fibrous actin, can be used for improving the shape of synapses, can improve repeated mechanical conduction of model mice suffering from autism diseases, takes effect quickly and is obvious in effects.

Description

technical field [0001] The invention belongs to the technical field of protein polypeptides, and in particular relates to a cortical actin mutant 8KQ and its application. Background technique [0002] Autism spectrum disorder (ASD) is a neurodevelopmental disorder that can be diagnosed clinically. The fifth edition of the latest "Diagnostic and Statistical Manual of Mental Disorders", the diagnostic criteria for ASD are summarized as: social communication disorder, repetitive stereotyped actions or interests, such as: rarely communicate with others, avoid the interlocutor's gaze, like to repeatedly rotate toys etc. In addition to the above-mentioned core symptoms, ASD patients may also show other atypical additional symptoms, such as varying degrees of intellectual disability, insomnia, anxiety, hyperactivity, and epilepsy. In 2000, the incidence rate of ASD was about 1-2‰. However, with the improvement of diagnostic standards and diagnostic methods, the incidence of ASD ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/47C12N15/12C12N7/01A61K38/17A61P25/00
CPCA61K38/00A61P25/00C07K14/4716C12N7/00C12N2740/15021C12N2750/14121
Inventor 朱筱娟马俊王雅君贺子轩何潇潇张禄卿郑耀武俞华莉申瑞雪
Owner NORTHEAST NORMAL UNIVERSITY
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