Project PXD014976

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Summary

Title

Arctigenin attenuates diabetic kidney disease through the activation of PP2A in podocytes

Description

Arctigenin (ATG) is a major component of Fructus Arctii, which as a traditional herbal remedy reduced proteinuria in diabetic patients. However, whether ATG specifically provided renoprotection in DKD was not known. Here we report that ATG administration is sufficient to attenuate proteinuria and podocyte injury in mouse models of diabetes. Transcriptomic analysis of diabetic mouse glomeruli showed that cell adhesion and inflammation are two key pathways affected by ATG treatment, and mass spectrometry analysis identified protein phosphatase 2A (PP2A) as one of the top ATG-interacting proteins in renal cells. Enhanced PP2A activity by ATG reduces p65 NF-κB-mediated inflammatory response and high glucose-induced migration in cultured podocytes via its interaction with Drebrin-1. Importantly, podocyte-specific Pp2a deletion in mice exacerbates DKD injury and abrogates the ATG-mediated renoprotection. Collectively, our results clearly demonstrate a renoprotective mechanism of ATG via PP2A activation and establish PP2A as a potential target for DKD progression.

Sample Processing Protocol

Mass spectrometry analysis: MS analysis was performed after DARTS or immunoprecipitation assay. Since these screening experiments were of n=1 sample, no statistical analysis was applied. In-solution digestion: The total protein concentration was determined using Branford Assay. Eight microgram of protein solutions (DMSO and ATG) from each sample was in-solution digested by trypsin. In brief, the protein was reduced by 2mM Dithiothreitol (DTT) at 55°C for 30 min followed by 10mM iodoacetamide (IAM) alkylation at room temperature for 10 min. Trypsin (Promega, Cat # V511A) was added in a ratio of 1:50 (trypsin:protein) and incubated at 37°C overnight. The resulted peptides were C18 desalted for LC-MS/MS analysis. In-gel digestion: The protein samples were separated on SDS-PAGE. After coomassie blue staining, the gel was cut into ~1mm cubes and rinsed with 30% acetonitrile (ACN) in 50 mM NH4HCO3 until the coomassie blue stain completely removed. 5 mM DTT was added to the gel for protein reduction at 55°C for 30 min followed by the alkylation with 25 mM IAM. Trypsin digestion was performed at 37°C for overnight. The resulting peptides were extracted and desalted with a C18 spin column (Thermo Fisher Scientific) for LC-MS/MS analysis. LC-MS/MS analysis: The peptides from either in-solution digestion or in-gel digestion were analyzed by LC-MS/MS on a U3000 Ultimate nano LC system coupled with Q Exactive MS instrument (Thermo Scientific). In brief, the peptides was resuspended in Solvent A (2% ACN in 0.1% FA) and separated on a C18 reversed phase nano column (Acclaim® Pep RSLC 75 μm × 50 cm, 2 μm, 100 Å, Thermo Scientific) with a 185-min of binary gradient of Solvent A and Solvent B (85% ACN in 0.1% FA). The gradient is set as the following: 0-10 min from 1%B to 5%B, 165 min to 30%B, 180 min to 50%B and 185 min to 95%B. The eluent peptides were direct introduced to mass spectrometry via Nanospray FlexTM ion source. The MS spectra was acquired in a positive mode with the spray voltage is 2.15 kV and the ion transfer tube temperature of 275 °C. The mass range is m/z 350-1700 with the resolution of 70,000 FWHM and AGC of 1E6 for MS scan. Fifteen most intensive ions with charge state between 2 and 5 were selected for MS/MS analysis with the dynamic exclusion of 60s. Higher-energy Collisional Dissociation (HCD) was use for peptide fragmentation with the Collison Energy of 27%. The resolution for MS/MS analysis was 17,500 FWHM with AGC of 5.0E4. Isolation window was set to 2 m/z and maximum injection time for both MS1 and MS2 were 100 ms.

Data Processing Protocol

Database Search: The MS/MS spectra were searched against a SwissProt human database (20,223 sequences) using a local MASCOT (V.2.3) search engine on the Proteome Discoverer (V1.4, PD) platform with the MS tolerance of 10 ppm and fragment tolerance of 0.1 Da. Carbomidomethylation of cysteine was selected as fixed modification, whereas oxidation of methionine, phosphorylation of serine and Threonine, and acetylation of N-terminus of protein were selected as variable modification. Maximum missed cleavage of trypsin is 2. The false discovery rate for both proteins and peptides was less than 1%. For the protein relative quantitation, the spectra counting method was used. To avoid exaggerating the ratio from the small spectra counts, we arbitrarily add 2 in each spectra counts prior to ratio calculation. Only the ratio changes over 2 folds were considered as changed. For phosphopeptides, the PhosphoRS node in PD was used to evaluate the probability of the phosphorylation sites assignment.

Contact

Tong Liu, Rutgers University
John Cijiang He, John Cijiang He, MD/PhD Division of Nephrology, Box 1243 Mount Sinai School of Medicine One Gustave L Levy Place, New York NY 10029 Tel: 212-659-1703, Fax: 212-987-0389 ( lab head )

Submission Date

12/08/2019

Publication Date

08/11/2019

Tissue

Not available

Experiment Type

Shotgun proteomics

Assay count

7

Publication

    Zhong Y, Lee K, Deng Y, Ma Y, Chen Y, Li X, Wei C, Yang S, Wang T, Wong NJ, Muwonge AN, Azeloglu EU, Zhang W, Das B, He JC, Liu R. Arctigenin attenuates diabetic kidney disease through the activation of PP2A in podocytes. Nat Commun. 2019 10(1):4523 PubMed: 31586053

Assay

Showing 1 - 7 of 7 results
# Accession Title Proteins Peptides Unique Peptides Spectra Identified Spectra View in Reactome
1 112258 RuiJie_Liu_ATG.mzid 2779 17980 10963 16695 0
2 112257 Ruijie_Liu_DMSO.mzid 2850 19948 12777 18241 0
3 112256 Ruijie_Liu_in-gel_EV_total.mzid 2094 10026 4682 7961 0
4 112255 Ruijie_Liu_in-gel_PP2a_total.mzid 2422 11026 5341 8688 0
5 112254 LiuRuijie_sample_ATG.mzid 3796 7613 3350 4712 0
6 112253 LiuRuijie_sample_DMSO.mzid 2950 5395 2447 3258 0
7 112252 LiuRuijie_sample_KD.mzid 3428 5769 2563 3350 0