Team:Nanjing high school/Engineering

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Engineering

Introduction

Microglia, as the resident immune cells of the central nervous system (Central nervous system, CNS), play an extremely important role in the brain. Under the stimulation of internal environment imbalance, microglia change from a resting state to an activated state, and the activated microglia secrete pro-inflammatory factors, glutamate, nitric oxide, etc., which intensify the inflammatory response and cause nerve damage. PPM1A (Protein phosphatase magnesium-dependent 1A), a protein phosphatase, can inhibit the production of inflammatory factors. PPM1A protein has anti-inflammatory ability in the body, can alleviate the inflammatory response that occurs after microglia is activated, prevent its long-term damage to nerves, and prevent microglia inflammation-related diseases, such as Alz Alzheimer's disease (Alzheimer's disease), Parkinson, etc.

Design

Recombinant human PPM1A protein was purified, and then was used for screen PPM1A agonists from a compound library (Figure 1). The anti-inflammatory ability of PPM1A agonist was tested on microglia.

Figure 1. The principle of PPM1A agonist screening. pNPP (p-nitrophenyl phosphate disodium salt) is used as a substrate. When the compound and PPM1A protein are added, the enzyme activity reaction will be initiated. The absorbance at 410 nm is measured with a microplate reader. By calculating the reaction rate, the agonistic efficiency of the compound was evaluated.

Build

DNA fragment coding PPM1A were synthesized and then inserted into expression vector pET28a. The construct was confirmed by DNA sequencing.

Test

Recombinant PPM1A was purified with a Ni-NTA column followed by AKTA FPLC purification. Then the purity of PPM1A was detected using SDS-PAGE (Figure 2).

Figure 2. SDS-PAGE analysis of expression and purification of PPM1A. The final yield recombinant protein was highlighted by red box.

PPM1A activator was screened in Lab in-house compound library by phosphatase enzyme activity assay with pNPP as the substrate. As indicated in Figure 3, among the compounds, compound 5 was finally selected for its highest enzymatic activity against PPM1A.

Figure 3. Screening of PPM1A activator by phosphatase enzyme activity assay.

The qPCR assay was further carried out to verify the inhibitive effect of compound 5 against PPM1A. BV-2 cells (a type of microglial cell derived from C57/BL6 murine) were co-incubated with LPS (Microglia will produce inflammatory factors such as IL-6 and TNFα under the stimulation of LPS) and different concentrations (5, 10, 20 μm) of compound 5 for 24 h. Then the mRNA level of IL-1β and IL-6 were detected by the qPCR assay. As shown in Figure 4, LPS effectively increased the mRNA level of IL-1β and IL-6, and compound 5 suppressed this increase effectively. Thus, these results confirmed that the suppressive effect of compound 5 against inflammation in BV-2 cells.

Figure 4. Compound 5 suppressed inflammation in microglial cells.

Learn

PPM1A enzyme was successfully produced by transformed Escherichia coli. On the basis of purified recombinant PPM1A, an excellent PPM1A activator was obtained. The compound, compound 5, enhanced PPM1A enzyme activity significantly. Our results implied that compound 5 was a PPM1A enzymatic activator. Compound 5 has good anti-inflammatory ability on microglia. Our results are promising to alleviate the inflammatory response that occurs after microglia are activated.

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