Dephosphorylation

Adding phosphate groups to intermediate metabolites

synonym Dephosphorylation (Dephosphorylation) generally refers to dephosphorylation

Dephosphorylation refers to the addition of phosphate groups to intermediate metabolites for discussion Alzheimer disease (AD) The possibility and pathway of brain injury reversal.

Chinese name: Dephosphorylation
Definition: Adding phosphate groups to intermediate metabolites

Project application: To explore the possibility and approach of brain injury reversal in Alzheimer's disease (AD)

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two method
three principle

topic

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discuss Alzheimer disease (AD) The possibility and pathway of brain injury reversal.

method

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Use dephosphorylation and Immunoblotting To study the reversibility of AD brain injury.

principle

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protein Phosphatase (PP) - 2A and PP-2B can dephosphorylate type II double helix (PHFII tau) in AD neurofibrillary tangles in Ser-199/Ser-202, and partially in Ser-396/Ser-404; In addition, PP-2A and PP-2B can dephosphorylate Ser-46 and Ser-235 of PHFII tau respectively; Relative of PHFII tau after dephosphorylation Electrophoretic mobility Accelerate. Mn2+and Mg2+can increase the dephosphorylation of PHFII tau by the above enzymes. The dephosphorylation of enzyme is concentration dependent, and the dephosphorylation increases with the increase of enzyme concentration.

conclusion

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because Tau protein Abnormal hyperphosphorylation and formation of PHF are considered to be the basis of neurofibrillar degeneration in AD, and the result that PHF can be dephosphorylated by protein phosphatase in vitro suggests that AD brain damage may be reversible.

There are three tau proteins in Alzheimer's (AD) brain: (1) soluble non abnormally phosphorylated tau (C-tau); (2) Soluble abnormal phosphorylated tau (ADp tau); (3) Tau (PHF tau) gathered by double helix. According to PHF tau at 2% Sodium lauryl sulfate (SDS) can also be divided into PHFI tau and PHFII tau [1]. Unlike ADp tau and PHFI tau, PHFII tau is also partially ubiquitinated [2].

According to Cohen [3]'s classification, a large number of phosphoserine and phosphothreonine proteins exist in human brain Phosphatase It mainly includes four types: PP-1, PP-2A, PP-2B and PP-2C. From previous studies, it was found that PP-2C did not dephosphorylate any site of ADp tau under study, while PP-1 only reacted to a few sites [4]. It is speculated that PP-2A and PP-2B may play a key role in the abnormal phosphorylation of tau in AD brain, so both of them were selected in this study.

Objects and Methods

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1、 Tissue and antibody source

The brain tissue of AD and age and sex matched controls in this study was derived from autopsy within 6 hours after death (6 cases were mixed to prepare homogenate). The diagnosis of AD was based on pathological examination of autopsy tissue sections. Human brain tissue was stored at - 75 ℃ until use. The separation and purification of bovine brain PP-2A and PP-2B refer to the methods of Cohen et al. [5] and Sharma et al. [6] respectively. Polyclonal antibody 92e and 102C were prepared by the Iqbal team. Monoclonal antibodies tau-1 and PHF-1 were provided by Binder and Greenberg respectively. Monoclonal antibodies SMI-31, SMI-33 and SMI-34 were purchased from Sternberg Monoclonal Company. alkaline phosphatase The labeled anti mouse and anti rabbit IgG was purchased from Sigma.

II Tau protein Sample preparation

PHFII tau protein was prepared from the brain of AD patients according to the long procedure of Iqbal et al. [7]. The simple steps are as follows: remove the meninges and white matter from the cerebral cortex of AD to prepare the homogenate, and then screen it with nylon membrane with different apertures SDS solution Medium heating and ultrasonic crushing, discontinuous Sucrose density gradient centrifugation And glass bead chromatography. For the preparation of ADP tau, refer to Kopke et al. [1].

3、 Dephosphorylation reaction

Unless otherwise indicated, the dephosphorylation reaction is carried out at 37 ℃ for 45 minutes. The reaction mixture contains 50mmol/LTris HCl (pH7.0), 20mmol/L β - mercaptoethanol ，0.1g/L bovine serum albumin ，1.0mmol/LMnCl2; 0.01g/L aprotinin, Leuppeptin and pepstatin, 0.07g/LPHF - Ⅱ - tau, 2U/ml PP-2A or 5U/ml PP-2B (1 unit PP-2A or PP-2B refers to the amount of enzyme that catalyzes phosphorylase a or phosphorylase kinase to release 1.0nmol phosphate per minute at 30 ℃ respectively). The dephosphorylation reaction solution with PP-2B also contains 1mmol/LCaCl2 and 1 μ mol/L calmodulin. The dephosphorylation reaction conditions of ADP tau are the same as those of PHFII tau, except for the lower phosphatase concentration [8, 9]. For detection of dephosphorylation sites Immunoblotting 。

4、 Immunoblotting

The sample after dephosphorylation treatment is first precipitated with 4 times volume of precooled acetone, and then dissolved in SDS-PAGE（ polyacrylamide gel electrophoresis ）Boil the sample buffer for 5 minutes, and then conduct 5%~15% SDS-PAGE. The dilution ratio and characteristics of the primary antibody used are shown in the attached table. For imprinting samples alkaline phosphatase Labeled anti mouse or anti rabbit IgG was used as the second antibody, and 5-bromo-4-chloro-3-indolylphosphate-p-aminotoluene salt (BCIP) and p-nitroblue tetrazolium chloride (NBT) were used as the substrates for color analysis.

result

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1、 PP-2A and PP-2B can dephosphorylate PHFII tau at multiple sites

PP-2A (2U/ml) and PP-2B (5U/ml) can color the tau-1 epitope of PHFII tau, and PP-2A and PP-2B can dephosphorylate Ser-46 and Ser-235 of PHFII tau, respectively. Both enzymes

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