ABSTRACT
Peptide drug discovery for the treatment of chronic kidney disease (CKD) has attracted much attention in recent years due to the urge to find novel drugs and mechanisms to delay the progression of the disease. In this study, we identified a novel short peptide (named YR-7, primary sequence 'YEVEDYR') from the natural Fibroin protein, and demonstrated that it significantly alleviated pathological renal changes in ADR-induced nephropathy. PANX1 was identified as the most notably upregulated component by RNA-sequencing. Further analysis showed that YR-7 alleviated the accumulation of lipid droplets via regulation of the lipid metabolism-related proteins PPAR α and PANK1. Using chemical proteomics, fluorescence polarization, microscale thermophoresis, surface plasmon resonance, and molecular docking, YR-7 was proven to directly bind to ß-barrel domains of TGM2 protein to inhibit lipid accumulation. TGM2 knockdown in vivo increased the protein levels of PPAR α and PANK1 while decreased the levels of fibrotic-related proteins to alleviate nephropathy. In vitro, overexpression TGM2 reversed the protective effects of YR-7. Co-immunoprecipitation indicated that TGM2 interacted with PANX1 to promote lipid deposition, and pharmacological inhibition or knockdown of PANX1 decreased the levels of PPAR α and PANK1 induced by ADR. Taken together, our findings revealed that TGM2-PANX1 interaction in promoting lipid deposition may be a new signaling in promoting ADR-induced nephropathy. And a novel natural peptide could ameliorate renal fibrosis through TGM2-PANX1-PPAR α/PANK1 pathway, which highlight the potential of it in the treatment of CKD.
ABSTRACT
BACKGROUND: Trefoil factor 1 (TFF1) mediates mucosal repair and belongs to a highly conserved trefoil factor family proteins which are secreted by epithelial cells in the stomach or colon mucous membrane. TFF1 forms a homodimer via a disulphide linkage that affects wound healing activity. Previous recombinant expressions of TFF1 were too low yield for industrial application. This study aims to improve the expression level of bioactive recombinant TFF1 (rTFF1) and facilitate application potency. METHODS: The rTFF1 gene rtff1 was synthesized, expressed by Escherichia coli and secreted by Brevibacillus choshinensis. The rTFF1s were purified. The polymeric patterns and wound healing capacities of purified rTFF1s were checked. RESULTS: In Escherichia coli, 21.08 mg/L rTFF1 was stably expressed as monomer, dimer and oligomer in soluble fraction. In Brevebacillus choshinensis, the rTFF1 was secreted extracellularly at high level (35.73 mg/L) and formed monomer, dimer and oligomer forms. Both proteins from different sources were purified by Ni-NTA chromatography and exhibited the wound healing activities. The rTFF1 produced by B. choshinensis had better wound healing capability than the rTFF1 produced by E. coli. After pH 2.4 buffer treatments, the purified rTFF1 formed more oligomeric forms as well as better wound healing capability. Glycosylation assay and LC-MS/MS spectrometry experiments showed that the rTFF1 produced by B. choshinensis was unexpectedly glycosylated at N-terminal Ser residue. The glycosylation may contribute to the better wound healing capacity. CONCLUSIONS: This study provides a potent tool of rTFF1 production to be applied in gastric damage protection and wound healing. The protein sources from B. choshinensis were more efficient than rTFF1 produced by E. coli.
