Marine polysaccharides have emerged as promising immunomodulators. However, conventional analytical approaches have failed to elucidate their activation mechanisms from a global perspective. In this study, we isolated a highly branched glycogen (GCEP2a) from eggs of the sea urchin Glyptocidaris crenularis . Structural characterization revealed that GCEP2a possesses a backbone composed of →4)-α-D-Glc p -(1 → residues, with one C6-branch per five residues, and a molecular weight of 8.06 × 10 3 kDa. Employing nascent-proteome bioorthogonal non-canonical amino acid tagging (BONCAT) enrichment followed by DIA-FAIMS, we unraveled the global immunostimulatory mechanisms of GCEP2a in RAW264.7 macrophages. This advanced approach enabled dynamic profiling of nascent protein synthesis, uncovering a multi-receptor and multi-pathway regulatory network. Specifically, GCEP2a activates signaling consistent with engagement of the toll-like receptors (TLRs), C-type lectin receptors (CLRs), and RIG-I-like receptors (RLRs), thereby triggering downstream signaling cascades including the NF-κB, MAPK, and PI3K-AKT pathways. Additionally, GCEP2a downregulates the biosynthesis of lacto- and neolacto-series glycosphingolipids, indicating metabolic reprogramming associated with M1 polarization. These findings not only suggest that GCEP2a exhibits potential as an immune adjuvant, but also provide initial insights into the application of BONCAT-based nascent proteomics for investigating the multi-modal immunostimulatory mechanisms of marine polysaccharides.
Tian Su,Zhen Wang,Zhengyi Zhang,Zhanwu Hou,Xiao Han,Fei Yang,Huadong Liu
Background Resveratrol, a well-known natural compound and nutrient, activates the deacetylation ability of SIRT1, demonstrating p53-dependent apoptosis functions in many diseases. However, the nascent proteomic fluctuation caused by resveratrol is still unclear. Objective In this study, we investigated the effect of resveratrol on the nascent proteome and transcriptome initiated by SIRT1 activation, and we explored the mechanism of resveratrol in HEK 293T cells. Methods Bioorthogonal noncanonical amino acid tagging (BONCAT) is a method used to metabolically label nascent proteins. In this strategy, L-azidohomoalanine (AHA) was used to replace methionine (Met) under different conditions. Taking advantage of the click reaction between AHA and terminal alkyne- and disulfide-functionalized agarose resin (TAD resin), we were able to efficiently separate stimulation responsive proteins from the pre-existing proteome. Resveratrol responsive proteins were identified by Liquid Chromatograph-Mass Spectrometer/Mass Spectrometer (LC-MS/MS). Furthermore, changes in mRNA levels were analyzed by transcriptome sequencing. Results Integrational analysis revealed a resveratrol response in HEK 293T cells and showed that Hsp60 was downregulated at both the nascent protein and mRNA levels. Knockdown of SIRT1 and Hsp60 provides evidence that resveratrol downregulated Hsp60 through SIRT1 and that Hsp60 decreased p53 through the Akt pathway. Conclusions This study revealed dynamic changes in the nascent proteome and transcriptome in response to resveratrol in HEK 293T cells and demonstrated that resveratrol downregulates Hsp60 by activating SIRT1, which may be a possible mechanism by which resveratrol prevents p53-dependent apoptosis by regulating Hsp60.
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