SIRT1, a highly conserved NAD+-dependent protein deacetylase, plays a pivotal role in the pathogenesis and therapy of atherosclerosis (AS). The aim of this study is to investigate the potential effects of SIRT1 on AS in ApoE–/– mice and the underlying mechanisms of autophagy in an ox-LDL-stimulated human monocyte cell line, THP-1. In vivo, the accelerated atherosclerotic progression of mice was established by carotid collar placement; then, mice were treated for 4 weeks with a SIRT1-specific inhibitor, EX-527. The atherosclerotic lesion size of EX-527-treated mice was greatly increased compared to that of the mice in the control group. Immunostaining protocols confirmed that the inhibition of SIRT1 during plaque initiation and progression enhanced the extent of intraplaque macrophage infiltration and impaired the autophagy process. In vitro cultured THP-1 macrophages exposed to ox-LDL were utilized to study the link between the SIRT1 function, autophagy flux, pro-inflammatory cytokine secretion, and foam cell formation using different methods. Our data showed that ox-LDL markedly suppressed SIRT1 protein expression and the autophagy level, while it elevated the MCP-1 production and lipid uptake. Additionally, the application of the SIRT1 inhibitor EX-527 or SIRT1 siRNA further attenuated ox-LDL-induced autophagy inhibition. In conclusion, our results show that the inhibition of SIRT1 promoted atherosclerotic plaque development in ApoE–/– mice by increasing the MCP-1 expression and macrophage accumulation. In particular, we demonstrate that blocking SIRT1 can exacerbate the acetylation of key autophagy machinery, the Atg5 protein, which further regulates the THP-1 macrophage-derived foam cell formation that is triggered by ox-LDL.
Hydroxytyrosol (HT), a phenolic compound in olive oil, exerts an anti-inflammatory effect in cardiovascular diseases. Recent studies found that autophagy was a therapeutic target of diseases. However, the effect of HT on autophagy in vascular adventitial fibroblasts (VAFs) remains unknown. Thus, in this study, we aimed to determine the effect of HT on cell autophagy and related signaling pathway and whether HT regulates the inflammatory response through autophagy in VAFs. Our results showed that HT promoted cell autophagy by increasing the conversion of LC3 and Beclin1 expression and the autophagic flux in VAFs stimulated with tumor necrosis factor-α (TNF-α). HT also upregulated the expression of the deacetylase sirtuin 1 (SIRT1) protein and mRNA compared with the TNF-α group. The molecular docking studies showed the good compatibility between HT and SIRT1, indicating that HT might act through SIRT1. Further study found that HT regulated autophagy through SIRT1-mediated Akt/mTOR suppression in VAFs. In addition, HT inhibited TNF-α-induced inflammatory response in VAFs through SIRT1. Furthermore, the study showed that HT inhibited the inflammatory response of VAFs through autophagy. These findings indicate that HT regulates the autophagy of VAFs through SIRT1-mediated Akt/mTOR suppression and then inhibits the inflammatory response of VAFs.
Many cellular functions necessitate structural assemblies of two or more associated proteins. The structural characterization of protein complexes using standard methods, such as X-ray crystallography, is challenging. Herein, we describe an orthogonal approach using hydrogen-deuterium-exchange mass spectrometry (HDXMS), cross-linking mass spectrometry (CXMS), and disulfide trapping to map interactions within protein complexes. HDXMS measures changes in solvent accessibility and hydrogen bonding upon complex formation; a decrease in HDX rate could account for newly formed intermolecular or intramolecular interactions. To distinguish between inter- and intramolecular interactions, we use a CXMS method to determine the position of direct interface regions by trapping intermolecular residues in close proximity to various cross-linkers (e.g., disuccinimidyl adipate (DSA)) of different lengths and reactive groups. Both MS-based experiments are performed on high-resolution mass spectrometers (e.g., an Orbitrap Elite hybrid mass spectrometer). The physiological relevance of the interactions identified through HDXMS and CXMS is investigated by transiently co-expressing cysteine mutant pairs, one mutant on each protein at the discovered interfaces, in an appropriate cell line, such as HEK293. Disulfide-trapped protein complexes are formed within cells spontaneously or are facilitated by addition of oxidation reagents such as HO or diamide. Western blotting analysis, in the presence and absence of reducing reagents, is used to determine whether the disulfide bonds are formed in the proposed complex interface in physiologically relevant milieus. The procedure described here requires 1-2 months. We demonstrate this approach using the β2-adrenergic receptor-β-arrestin1 complex as the model system.
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