What Are Phosphorylation Sites?

Dephosphorylation refers to the addition of phosphate groups to intermediate metabolites, and is used to explore the possibility and ways of reversing Alzheimer's disease (AD) brain damage.

Dephosphorylation

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Dephosphorylation refers to the addition of phosphate groups to intermediate metabolites, and is used to explore the possibility and ways of reversing Alzheimer's disease (AD) brain damage.

Chinese name

Dephosphorylation

Definition

Add phosphate groups to intermediate metabolites

To explore the possibility and ways of reversing Alzheimer's disease (AD) brain injury.

Dephosphorylation and immunoblotting were used to study the reversibility of AD brain injury.

Protein phosphatase (PP) -2A and PP-2B can dephosphorylate type II double helix (PHFII-tau) in AD neurofibrillary tangles at Ser-199 / Ser-202, Ser-396 / Ser -404 is partially dephosphorylated. In addition, PP-2A and PP-2B can dephosphorylate Ser-46 and Ser-235 of PHFII-tau, respectively; the relative electrophoretic mobility of PHFII-tau is accelerated after dephosphorylation. Mn2 + and Mg2 + can increase the dephosphorylation of PHFII-tau by these enzymes. The dephosphorylation of the enzyme is concentration-dependent. As the enzyme concentration increases, the dephosphorylation increases.

Because abnormally hyperphosphorylation of tau protein and the formation of PHF are considered to be the basis of AD neurofibrillary degradation, the results that PHF can be dephosphorylated by protein phosphatase in vitro suggest that AD brain damage may be reversible.

There are three types of tau proteins in Alzheimer's (AD) brain: (1) soluble and non-abnormally phosphorylated tau (C-tau); (2) soluble and abnormally phosphorylated tau (ADp-tau); ( 3) tau (PHF-tau) aggregated in double helix. According to the solubility of PHF-tau in 2% sodium lauryl sulfate (SDS), it can be divided into PHFI-tau and PHFII-tau [1]. Unlike ADp-tau and PHFI-tau, PHFII-tau is also modified by partial ubiquitination [2].

According to the classification of Cohen [3], a large number of phosphoseyl and phosphothreonyl protein phosphatases present in human brain mainly include four types: PP-1, PP-2A, PP-2B, and PP-2C . It has been found from previous studies that PP-2C does not dephosphorylate any site of ADp-tau studied, while PP-1 only reacts 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 were selected for this study.

I. Tissue and antibody sources

The brain tissues of AD and age- and sex-matched controls in this study were derived from autopsies within 6 hours after death (6 patients were each mixed and homogenized). The diagnosis of AD was based on pathological examination of autopsy tissue sections and human brain. Tissues were stored at -75 ° C until use. For the isolation 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 antibodies 92e and 102C were prepared by the Iqbal group. 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-labeled anti-mouse and anti-rabbit IgG were purchased from Sigma.

Preparation of tau protein samples

PHFII-tau protein was prepared from the brain of AD patients according to the long procedure of Iqbal et al. [7]. The brief steps are as follows: remove the meninges and white matter from AD cerebral cortex, prepare homogenate, then sieving with nylon membranes with different pore sizes, heat and sonicate in 2% SDS solution, discontinuous sucrose density gradient centrifugation and glass bead chromatography . For the preparation of ADP-tau, see Kopke et al. [1].

Third, the dephosphorylation reaction

Unless otherwise specified, dephosphorylation reactions were performed at 37 ° C for 45 minutes. The reaction mixture contains 50mmol / LTris-HCl (pH7.0), 20mmol / L -mercaptoethanol, 0.1g / L bovine serum albumin, 1.0mmol / LMnCl2; each 0.01g / L aprotinin, Leupeptin And pepsstatin, 0.07g / LPHF--tau, 2U / mlPP-2A or 5U / mlPP-2B (1 unit of PP-2A or PP-2B refers to catalytic phosphorylation per minute at 30 ° C, respectively Enzyme a or phosphorylase kinase releases an enzyme amount of 1.0 nmol of phosphate). The dephosphorylation reaction solution using PP-2B also contained 1 mmol / LCaCl2 and 1 mol / L calmodulin. ADP-tau dephosphorylation reaction conditions are the same as PHFII-tau dephosphorylation conditions except that lower phosphatase concentrations are used [8, 9]. Dephosphorylation sites were detected by immunoblotting.

Fourth, immunoblotting

The dephosphorylated sample was first precipitated with 4 times the volume of pre-cooled acetone, and then dissolved in the SDS-PAGE (polyacrylamide gel electrophoresis) sample buffer. After boiling for 5 minutes, 5% to 15% SDS-PAGE was performed. The dilution factors and characteristics of the primary antibodies used are shown in the attached table. Blotting samples used alkaline phosphatase-labeled anti-mouse or anti-rabbit IgG as secondary antibodies, 5-bromo-4-chloro-3indole phosphate-p-aminotoluene salt (BCIP) and p-nitroblue tetrazole chloride ( NBT) was used as a substrate for color development analysis.

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

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