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Cortex actin mutant 8KQ and application

An actin and mutant technology, applied in the field of protein and peptides, can solve the problems of restricting the research on the pathogenesis of ASD and the development of clinical drugs, and achieve the effects of improving repetitive stereotyped behavior, improving synaptic morphology, and reducing binding capacity.

Active Publication Date: 2019-04-12
NORTHEAST NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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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  • Cortex actin mutant 8KQ and application
  • Cortex actin mutant 8KQ and application
  • Cortex actin mutant 8KQ and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Excitatory synapses express acetylated cortical actin

[0038] Coat petri dishes or slides with polylysine (PLL) in advance, and coat overnight at room temperature or coat at 37°C for 2 hours. Recover PLL, rinse with sterile water 3 times, and store the coated petri dish or glass slide at 4°C for 1 week. Pre-cool HBSS buffer, and pay attention to the operation before digestion to maintain low temperature and sterility. The suckling mouse was stunned on the day of birth, disinfected with 75% alcohol, and the brain was quickly taken. Strip the hippocampus / cortex tissue in HBSS and gently transfer to EP tube containing fresh HBSS. Cut into small pieces, digest with trypsin (0.125%) at 37°C for 30 min, during which the tissue pieces are gently resuspended every 10 min. 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 resuspend, and let stand at 37°C for 5 min. Use ...

Embodiment 2

[0042] Studies have shown that the acetylation of cortical actin is regulated by acetyltransferase (PCAF) and deacetylase (HDAC6), and non-enzymatic acetylation may also exist. Acetyl-CoA (Ac-CoA) is the only source of acetyl groups required for protein acetylation. The concentration of Ac-CoA in the cell affects the level of acetylation modification of the protein. To explore this possibility and its biological significance, an in vitro acetylation experiment was carried out. Reaction buffer: Tris-HCl (50mM, pH8.0), EDTA (0.1mM), DTT (1mM), glycerol (10%), sodium butyrate (10mM), add protease inhibitor before use. Reaction substrate: cortical actin, acetyl donor Ac-CoA. The reaction system was incubated at 30°C for 1 hour, and the resulting reaction product was used for Western blot antibody detection. The antibody used was a pan-acetylated antibody acetylated-lysine; the amount of substrate cortical actin added was indicated by Coomassie brilliant blue staining. In the in v...

Embodiment 3

[0044] Identification of the acetylation site of cortical actin

[0045] In order to further explore the role of cortical actin acetylation in the regulation of postsynaptic structure and function, the LC-MS / MS method was used to identify in vitro acetylation experiments ( figure 2 ) The acetylation site of actin in the middle cortex. The SDS strips at the location of cortical actin stained with Coomassie brilliant blue were cut off, and the subsequent LC-MS / MS process was completed by Hangzhou Jingjie Biotechnology Co., Ltd. The specific process is as follows: the glue strip is cut into pieces, and the glue block is decolorized with 50% acetonitrile containing 50 mM ammonium bicarbonate (NH4HCO3). Incubate with 100% acetonitrile for 5 min to dehydrate the gel block, then remove the liquid phase from the system, add a dithiothreitol solution with a final concentration of 10 mM, and incubate at 37° C. for 60 min. Incubate with 100% acetonitrile again for dehydration, remove the ...

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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 and polypeptide, and particularly relates to a cortical actin mutant 8KQ and its application. Background technique [0002] Autism spectrum disorder (ASD) is a type of neurodevelopmental disease that can be diagnosed by clinical manifestations. The latest "Diagnosis and Statistics Manual of Mental Disorders" fifth edition, the diagnostic criteria for ASD are summarized as: social communication disorder, repetitive stereotyped actions or interests, such as: rarely communicate with others, avoid the eyes of the interlocutor, and like to rotate repeatedly Toys etc. In addition to the above-mentioned core symptoms, ASD patients may also show other atypical additional symptoms, such as varying degrees of mental retardation, insomnia, anxiety, hyperactivity, epilepsy, etc. The incidence of ASD in 2000 was about 1 to 2‰. However, with the improvement of diagnostic criteria and diagnostic methods, the incid...

Claims

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Application Information

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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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