Cryptotanshinone Targets HYOU1 to Rewire ER-Mitochondria Communication and Enhance Autophagy in Atherosclerosis

Cryptotanshinone Targets HYOU1 to Rewire ER-Mitochondria Communication and Enhance Autophagy in Atherosclerosis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Cryptotanshinone Targets HYOU1 to Rewire ER-Mitochondria Communication and Enhance Autophagy in Atherosclerosis Minjeong Ko, Jungmin Ha, Sunyoung Kwon, Hye Eun Lee, Ji Young Mun, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6301868/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Atherosclerosis is driven by chronic lipid accumulation, oxidative stress, and impaired autophagy, yet effective therapies targeting these pathways remain elusive. Oxidized low-density lipoprotein (oxLDL), a major contributor to atherosclerosis, disrupts cellular homeostasis by promoting the formation of mitochondria-associated ER membrane (MAM), which regulate essential processes such as calcium transport, lipid metabolism, mitochondrial dynamics, and autophagy—critical components in atherosclerotic signaling pathways. Here, we identify hypoxia-upregulated protein 1 (HYOU1) as a novel regulator of MAM stability, coordinating ER-mitochondrial calcium flux and lipid metabolism. Using DARTS-LC-MS/MS, we demonstrate that cryptotanshinone (CTS), a natural anti-atherosclerotic compound derived from Danshen ( Salvia miltiorrhiza ), binds directly to the nucleotide-binding domain (NBD) of HYOU1, disrupting ER-mitochondria interactions. This results in three key outcomes: (1) increased cytosolic calcium levels and TFEB nuclear translocation, (2) decreased mitochondrial calcium influx and ATP production, and (3) enhanced autophagy, which reduces intracellular lipid accumulation. Consequently, CTS reduces mitochondrial ROS accumulation and lipid overload in vitro , while alleviating atherosclerotic burden in ApoE-/- mice fed a high-cholesterol diet. Single-cell transcriptome analysis of human carotid atherosclerotic plaques further revealed that HYOU1 and its associated autophagy and lipid metabolism regulators are upregulated in monocytes and dendritic cells, underscoring their clinical relevance and co-regulated expression in atherosclerotic lesions. These findings provide the first evidence that HYOU1 as a promising therapeutic target for modulating MAM-autophagy crosstalk and highlight CTS as a potential small-molecule intervention to disrupt the oxLDL-driven atherosclerotic cycle. Cell Communication and Signaling Atherosclerosis Mitochondria-Associated ER Membranes (MAM) Hypoxia-Upregulated Protein 1 (HYOU1) Cryptotanshinone (CTS) Autophagy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Cardiovascular disease (CVD) remains the leading cause of mortality worldwide, with atherosclerosis being a primary contributor. Atherosclerosis is driven by lipid accumulation, chronic inflammation, and endothelial dysfunction, ultimately leading to plaque formation and vascular complications. 1 Oxidized low-density lipoprotein (oxLDL), a key driver of atherosclerosis, induces oxidative stress, endothelial damage, and macrophage activation, accelerating plaque formation. 2 Lipid accumulation in atherosclerosis triggers cellular stress including endoplasmic reticulum (ER) stress, disrupting signaling pathways that drive disease progression. 3 Beyond these well-known effects, oxLDL also perturbs intracellular homeostasis by enhancing the formation of mitochondria-associated ER membranes (MAMs), leading to excessive mitochondrial calcium influx and impaired autophagy. 4 , 5 Given that autophagy is crucial for lipid clearance and inflammation resolution, its dysregulation, combined with increased mitochondrial reactive oxygen species (mROS) production, establishes a pathological loop that accelerates atherosclerosis progression. We define this feedback mechanism as the 'oxLDL-driven atherosclerotic cycle,' where oxLDL-induced MAM formation suppresses autophagy and promoting excessive mROS generation (Fig. 1 a). Elevated mROS further drive oxidation of native LDL, leading to increased oxLDL accumulation, thereby reinforcing the cycle. Organelle crosstalk plays a fundamental role in maintaining cellular homeostasis, with MAM serving as a critical interface for ER- mitochondria interactions. 6 , 7 The MAM regulates key processes such as calcium transport, lipid metabolism, mitochondrial dynamics, and autophagy. 8 One of the major protein complexes, the IP 3 R-Grp75-VDAC1 complex, facilitates calcium flux between the ER and mitochondria. 9 Loss of MAM integrity due to impaired VAPB (ER protein) and PTPIP51 (mitochondrial protein) disrupt calcium exchange and subsequently induces autophagy. 10 Furthermore, mitofusin-2 (MFN2), a mitochondrial fusion protein, links the ER and mitochondria, interacting with AMPK to regulate autophagy under metabolic stress. 11 , 12 Despite growing evidence that MAM integrity is essential for maintaining metabolic balance, therapeutic strategies targeting MAM in atherosclerosis remain largely unexplored. While MAM dysfunction has been extensively studied in neurodegenerative and metabolic disorders, its role in cardiovascular diseases, particularly atherosclerosis, remains poorly understood. Natural compounds have gained attention for their potential to modulate key signaling pathways, including those governing metabolism and organelle dynamics. 13 – 15 Cryptotanshinone (CTS), a bioactive component of Danshen ( Salvia miltiorrhiza ), has demonstrated anti-atherosclerotic effects. 16 , 17 However, its impact on MAM dynamics and molecular targets remains unexplored. Identifying the cellular targets of CTS could provide new insights into regulatory pathways governing organelle crosstalk and autophagy. In this study, we investigate the potential of Hypoxia Up-regulated Protein 1 (HYOU1, also known as GRP170) as a target for CTS-mediated modulation of ER-mitochondrial interactions. HYOU1 is an ER-resident chaperone involved in protein quality control and the regulation of the unfolded protein response (UPR), which is activated by ER stress. 18 HYOU1 contains a nucleotide-binding domain (NBD) that mediates its chaperone activity through ATP/ADP binding. 19 Although HYOU1’s role in atherosclerosis has been underexplored, recent studies have shown that targeting its NBD can suppress pathogenic fibroblast activity. 20 Given that HYOU1 may regulate MAM integrity and metabolic stress, targeting its NBD could represent a therapeutic approach for modulating ER-mitochondrial crosstalk in atherosclerosis. Here, we explore the role of MAM integrity and autophagy in atherosclerosis and examine whether targeting this pathway could provide a therapeutic strategy. We assess the effects of pharmacological modulation of MAM and investigate the contribution of HYOU1 to metabolic stress in atherosclerosis. These findings may offer new insights into the molecular mechanisms linking ER-mitochondrial crosstalk, autophagy, and lipid accumulation, providing a potential framework for therapeutic strategies targeting organelle communication in cardiovascular disease. Results CTS Regulates ER-Mitochondria Interactions to Mitigate oxLDL-Induced Cellular Dysfunction We define the ‘ox-LDL driven atherosclerotic cycle’ as the feedback loop initiated by oxLDL, which triggers the formation of MAMs, suppresses autophagy, and induces cellular stress ( Fig. 1a ). This cycle exacerbates atherosclerosis progression by disrupting cellular homeostasis. To explore the impact of oxLDL on cellular physiology, we treated HUVECs with 50 μg/mL oxLDL, which inhibited cell proliferation at both 24 and 48 hours ( Supplementary Fig. 1a ). We further confirmed that oxLDL enhances ER-mitochondrial interactions, demonstrated by GFP fluorescence complementation using the SPLICS system ( Supplementary Fig. 1b ), 21 and proximity ligation assay (PLA), a technique that detects protein-protein interactions within close proximity, applied to IP 3 R-VDAC1 pairs ( Supplementary Fig. 1c ). This interaction led to elevated mitochondrial calcium levels, a response that could be blocked by the IP 3 R antagonist xestospongin C, highlighting a role for IP 3 R in ER-mitochondrial calcium transport ( Supplementary Fig. 1d ). Additionally, oxLDL induced modest ER-lysosome interactions but did not significantly affect mitochondria-lysosome interactions ( Supplementary Fig. 1e-f ). Importantly, oxLDL treatment impaired autophagic flux, as evidenced by a decrease in TFEB nuclear translocation and the accumulation of SQSTM1, LC3-II, and autophagosomes ( Supplementary Fig. 1g-i ). These impairments were associated with increased expression of CHOP, a marker of ER stress ( Supplementary Fig. 1j-k ). 22 oxLDL treatment also disrupted mitochondrial function, leading to elevated mitochondrial ROS (mROS) levels, swelling, and fragmentation ( Supplementary Fig. 1l-m ). These findings highlight the role of the atherosclerotic cycle in exacerbating cellular dysfunction driven by oxLDL. We explore potential therapeutic approaches aimed at disrupting this cycle to mitigate the pathological effects associated with atherosclerosis. Our previous study indicated that a Salvia miltiorrhiza (Danshen) extract reduced atherosclerosis via autophagy. 17 We investigated the active compounds of Danshen for their ability to inhibit foam cell formation, a hallmark of atherosclerosis, in oxLDL-stimulated RAW264.7 macrophages. Among the major lipophilic compounds, cryptotanshinone (CTS) exhibited superior inhibitory activity on foam cell formation compared to tanshinone-II-A (Tan II A) and dihydrotanshinone-I (DHT) ( Fig. 1b ). Next, we examined the effects of CTS on oxLDL-induced MAM dynamics in HUVECs. An MTT assay revealed that the IC 50 of CTS was approximately 5 µM in HUVECs ( Supplementary Fig. 2a ). Using transmission electron microscopy (TEM), we observed that 4-hour CTS treatment decreased ER-mitochondria contact points within 40 nm, from 61% to 37%, in oxLDL-treated HUVECs, without altering mitochondrial area or circularity ( Fig. 1c , Supplementary Fig. 2b-d ). CTS treatment reduced mitochondrial swelling and the oxLDL-induced increase in ER-mitochondria contact area ( Fig. 1c ). PLA experiments revealed that oxLDL treatment increased IP 3 R-VDAC1 interactions, which were reduced upon CTS treatment ( Fig. 1d ). Importantly, CTS did not affect the expression levels of IP 3 R or VDAC1 within the 4 hours of treatment ( Supplementary Fig. 2e ). To assess microtubule involvement, cells were treated with tubulin polymerization inhibitors (taxol and vinblastine). CTS induced a greater reduction in ER-mitochondria contacts than these inhibitors ( Supplementary Fig. 2f ), indicating that it does not directly disrupt microtubule dynamics at early time points. MAM is crucial for the transport of calcium and lipids between the ER and mitochondria. To assess the impact of CTS on ER-mitochondrial communication, we first evaluated mitochondrial calcium dynamics. Using Rhod-2 fluorescence to monitor mitochondrial calcium, we found that histamine-induced calcium influx (via IP 3 R activation) increased mitochondrial calcium levels in oxLDL-treated cells, which was associated with enhanced ER-mitochondrial interactions. However, this response was significantly reduced in CTS-treated cells, suggesting that CTS disrupts calcium transfer by altering ER-mitochondrial proximity ( Fig. 1e ). Notably, CTS did not alter p-IP 3 R levels, indicating that ER calcium release was unchanged ( Supplementary Fig. 2g ). 23 The reduced mitochondrial calcium levels in CTS-treated cells corresponded to a decrease in ATP production ( Fig. 1f ). Furthermore, lipid transport between the ER and mitochondria was assessed using nUHPLC-ESI-MS/MS. CTS treatment decreased mitochondrial levels of several phospholipids, including phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylethanolamine (PE), and phosphatidylcholine (PC), suggesting altered lipid transfer between the two organelles ( Fig. 1g ). Holotomography is an advanced imaging technology that captures 3D images of live cells without fluorescence staining and analyzes various cellular parameters based on refractive index measurements. 24 To assess the impact of CTS on intracellular dynamics, we used holotomography to visualize HUVECs after 24 hours of treatment with oxLDL or oxLDL+CTS. This analysis revealed mitochondrial fragmentation and the accumulation of globular, refractile structures indicative of lipid in oxLDL-treated cells, both of which were alleviated by CTS treatment ( Fig. 1h ). AI-based analysis revealed that oxLDL-treated cells formed distinct clusters, whereas oxLDL+CTS-treated cells exhibited clustering patterns similar to those of control cells depending on the duration of treatment effectively alleviating the morphological changes induced by oxLDL ( Fig. 1i ). Furthermore, in the presence of oxLDL, CTS treatment reduced ER stress and in CHOP-mCherry reporter cells 22 ( Fig. 1j, Supplementary Fig. 2h ) and mROS in HUVECs ( Fig. 1k ), leading to improved cellular health and alleviation of pathological factors associated with atherosclerosis. CTS Enhances Autophagic Flux in Atherosclerosis Lysotracker fluorescence intensity increased by approximately 20% in CTS-treated cells, suggesting the restoration of lysosomal function impaired by oxLDL treatment ( Fig. 2a, Supplementary Fig.3a ). CTS treatment led to an increase in LC3-I to LC3-II conversion levels and promoted both the accumulation and degradation of SQSTM1 over time ( Fig. 2b ). Co-treatment with CTS and chloroquine (CQ, an autophagy inhibitor), further elevated LC3-II levels compared to CQ alone, demonstrating CTS-induced autophagic flux ( Fig. 2c ). Since calcium signaling is crucial for autophagy regulation and ER-mitochondria interactions maintain calcium homeostasis, we next focused on cytoplasmic calcium dynamics to explore how reduced ER-mitochondria interactions affect intracellular signaling. In CTS-treated HUVECs, cytosolic calcium levels increased, as shown by Fluo-4 fluorescence ( Fig. 2d ). This increase was not due to lysosomal calcium release, as confirmed by the use of GCaMP3-TRPML1, a genetically encoded calcium sensor specifically targeting lysosomes. 25 GCaMP3 fluorescence remains unchanged with CTS treatment ( Supplementary Fig. 3b ), confirming that CTS alters cytosolic calcium dynamics independently of lysosomal release. Disruption of MAM and the subsequent elevation in cytosolic calcium by CTS induced dose-dependent nuclear translocation of TFEB, as confirmed by immunoblotting and immunofluorescence staining ( Fig. 2e, Supplementary Fig. 3c ). These findings suggest that CTS treatment promotes autophagic flux through the disruption of the ER-mitochondria interactions, which in turn modulates calcium signaling pathways critical for autophagy regulation, including the activation of TFEB and downstream autophagic markers. To evaluate the lipid-lowering effects of CTS, DiI-labeled oxLDL-treated HUVECs were analyzed. Co-treatment with bafilomycin A1 (BafA1), an autophagy inhibitor, blocked CTS-mediated lipid reduction, confirming that this process is autophagy-dependent ( Fig. 2f ). Moreover, CTS significantly reduced lipid levels in wild-type HeLa cells, but had no effect in ATG3 knockout HeLa cells, which are defective in autophagosome formation ( Fig. 2g-h ). These findings suggest that CTS could serve as a potential therapeutic agent in treating atherosclerosis and other metabolic disorders by enhancing autophagic flux and reducing lipid accumulation in endothelial cells. CTS Ameliorates Atherosclerosis in ApoE-/- Mice via Autophagy Induction We next evaluated the efficacy of CTS in modulating autophagy and alleviating atherosclerosis in an ApoE-/- mouse model. CTS (20 mg/kg) was administered intraperitoneally every 2 days for 8 weeks to mice fed a high-cholesterol diet ( Fig. 3a ). This treatment did not affect body weight ( Fig. 3b ), indicating low toxicity. Notably, serum total cholesterol and triglyceride levels decreased in CTS-treated mice ( Fig. 3c-d ), and Oil-Red-O staining revealed a reduction in the aortic root lesion area ( Fig. 3e ). Furthermore, atherosclerotic plaque formation in the aorta was significantly reduced in CTS-treated mice compared to vehicle-treated controls ( Fig. 3f ). We then investigated whether CTS induces autophagy and modulates MAM in vivo. LC3B and SQSTM1 colocalization was assessed in aortic sinus sections, revealing increased intensities of both markers in CTS-treated mice ( Supplementary Fig. 3d ). In primary mouse peritoneal macrophages, CTS treatment reversed the decrease in LC3B levels and the increase in lipid content observed in vehicle-treated animals ( Fig. 3g ). Additionally, PLA assays between IP 3 R and VDAC1 on aortic sinus sections revealed a marked reduction in PLA dot numbers, indicating decreased ER-mitochondria interactions following CTS treatment ( Fig. 3h ). Similarly, PLA performed on peritoneal macrophages of CTS-treated mice showed a decrease in PLA dot numbers ( Fig. 3i ). These findings suggest that CTS, through the modulation of autophagy and disruption of ER-mitochondrial interactions, holds promise as a targeted therapeutic strategy to alleviate atherosclerosis by reducing lipid accumulation and plaque formation in vascular tissues. Identification of HYOU1 as a Target of CTS Drug affinity responsive target stability (DARTS)-LC-MS/MS is a powerful technique for identifying small molecule targets without requiring chemical modification, 26, 27 and has been previously employed to identify targets of autophagy-regulating compounds. 28-30 Using this approach, we aimed to identify CTS target proteins involved in ER-mitochondria interactions and autophagy, with a particular focus on ER-localized proteins due to their central role in stress response and autophagy regulation. Membrane proteomes were treated with CTS and analyzed by DARTS-LC-MS/MS ( Fig. 4a ). SWATH analysis quantified 872 proteins, with 329 exhibiting a coefficient of variation (CV) of less than 20%. Among these, 69 proteins were degraded by pronase (>10%), while 16 proteins were stabilized by CTS binding (FC ≥ 1.2, Fig. 4b ). Candidate proteins were predominantly localized to the ER or mitochondria ( Fig. 4c ), with STRING analysis linking them to key processes including ER stress (HYOU1 and CANX), vesicle trafficking (RAB1A and RAB10), and metabolism (ALDH18A1, HSD17B10, and DLAT) ( Fig. 4d ). Our focus was on ER-localized candidates ( Fig. 4e ), given the ER's central role in stress response and autophagy regulation. DARTS-Western blot analysis of HUVECs lysates confirmed that HYOU1 showed resistance to degradation by pronase in the presence of CTS binding, demonstrating a clear binding pattern. In contrast, RAB10 and CANX did not show a similar pattern, further supporting the specificity of CTS-HYOU1 interaction ( Supplementary Fig. 4a-b ). LC-MS/MS analysis identified 16 peptide fragments corresponding to HYOU1, along with 7 peptides in the NBD and SBD domains, which exhibited reduced degradation upon pronase treatment ( Supplementary Fig. 4c ). Dose-dependent DARTS analysis revealed an EC50 of approximately 1 µM for CTS binding to HYOU1 ( Fig. 4f ). Further validation via a cellular thermal shift assay (CETSA) demonstrated that CTS prevented thermal denaturation of HYOU1 in live HEK293 cells ( Fig. 4g ). Full-length HYOU1 constructs were cloned into the pEZT-BM vector 31 and the HYOU1(1-912) protein was expressed and purified ( Supplementary Fig. 4d ). Microscale thermophoresis (MST) analysis of CTS binding to HYOU1 showed a dissociation constant (Kd) of 1.42 µM, confirming direct interaction between CTS and HYOU1 ( Fig. 4h ). Identification of Critical Binding Sites for CTS on HYOU1 To investigate potential binding sites for CTS on HYOU1, we employed in silico docking analysis using the Alphafold 3D structure of HYOU1 ( Fig. 5a ). CTS was predicted to bind a pocket within the NBD of HYOU1, with residues R217 and N410 likely interacting with CTS via hydrogen bonds ( Fig. 5b ). We experimentally verified these binding sites by generating point mutations: HYOU1-R217A and N410D. DARTS analysis of HEK293 cells transfected with MYC-tagged wild-type (WT) or mutant HYOU1 plasmids revealed that CTS could not protect the mutant HYOU1 from proteolysis, confirming the critical role of these residues in CTS binding ( Fig. 5c ). Moreover, in HUVECs expressing mutant HYOU1, CTS treatment led to reduced autophagosome formation, as assessed by LC3B immunofluorescence staining ( Fig. 5d ), highlighting the importance of R217 and N410 in CTS-mediated autophagy regulation. Collectively, we identified HYOU1 as a target of CTS through DARTS-LC-MS/MS and validated this interaction using in silico and in vitro assays. HYOU1 as a Key Mediator of CTS-Regulated ER-Mitochondrial Interactions and Autophagy Glucose-regulated proteins (GRPs), localized in the ER and mitochondria, function as chaperones that maintain organelle homeostasis. 18 Specifically, GRP75 forms the IP 3 R-GRP75-VDAC1 complex, contributing to ER-mitochondrial coupling, while GRP78 interacts with IP 3 R1 to facilitate ER calcium release. 32 Although HYOU1 (also known as GRP170) shares structural and functional similarities with GRP75 and GRP78, 18 it has not been as extensively studied. Therefore, we hypothesized that HYOU1 may play a distinct role in regulating MAM and autophagy. We examined how CTS-mediated regulation of HYOU1 affects ER-mitochondria interactions. We speculated that HYOU1 might indirectly modulate the IP 3 R-GRP75-VDAC1 complex, potentially involving GRP78 in this interaction network ( Fig. 6c ). PLA results showed that CTS treatment for 4 hours reduced interactions between HYOU1-IP 3 R, HYOU1-GRP78, and IP 3 R-GRP78, which led to the dissociation of IP 3 R-GRP75, GRP75-VDAC1, and IP 3 R-VDAC1 complexes ( Fig. 6a ). Co-immunoprecipitation further confirmed the reduced interaction between HYOU1 and IP 3 R in CTS-treated cells ( Fig. 6b ), suggesting that CTS binding induces a conformational change in HYOU1, decreasing its affinity for IP 3 R ( Fig. 6c ). A recently developed HYOU1 inhibitor (HYOU1-IN), 20 intended for fibroblast activation suppression, was found to modulate MAM dynamics ( Supplementary Fig. 5a-b ). In addition, HYOU1-IN disrupted the interaction between HYOU1 and GRP78 ( Supplementary Fig. 5c ), suggesting that this interaction is critical for MAM complex stability. Notably, CTS exhibited greater potency than HYOU1-IN in modulating MAM dynamics. These findings demonstrate a functional link between HYOU1 and MAM homeostasis, providing mechanistic insights into its role in disease progression and underscoring its potential as a therapeutic target. We next examined whether HYOU1 mediates the biological effects of CTS by assessing MAM formation following HYOU1 knockdown. In HYOU1-depleted HUVECs, ER-mitochondria contacts were disrupted ( Fig. 6d ), cytosolic calcium levels increased ( Fig. 6e ), and autophagic flux was enhanced, as indicated by increased LC3-II conversion and reduced SQSTM1 ( Fig. 6f ). Additionally, lipid levels were reduced in these cells ( Fig. 6g ), and oxLDL-induced foam cell formation was inhibited in HYOU1-deficient macrophages ( Fig. 6h ), suggesting a role for HYOU1 in atherosclerosis progression. To confirm that HYOU1 is a critical target of CTS, we generated HYOU1 heterozygous knockout (KO) cells using CRISPR-Cas9 ( Fig. 7a, Supplementary Fig. 6a-b ). 33, 34 PLA assays revealed diminished ER-mitochondria contact in HYOU1 KO cells compared to WT cells ( Fig. 7b ), indicating that HYOU1 is essential for maintaining MAM integrity. Upon CTS treatment, the effects on ER-mitochondria contacts ( Fig. 7c-d ), lipid levels ( Fig. 7e ), and other CTS-induced changes were reduced in HYOU1 KO cells compared to WT cells ( Supplementary Fig. 6c-d ). Association of HYOU1 with Atherosclerosis Progression To assess the relevance of our mechanistic findings in human disease, we analyzed single-cell RNA sequencing (scRNA-seq) data from human carotid atherosclerotic plaques (AC) and adjacent tissue (PA) from patients undergoing carotid endarterectomy (GSE159677; n=3 per group). 35 Unsupervised clustering identified 10 major cell populations, including monocytes, dendritic cells, T cells, endothelial cells, smooth muscle cells, fibroblasts, B cells, plasma cells, mast cells, and NK cells ( Fig. 7f ). We visualized gene expression across these clusters using FeaturePlot analysis. Genes involved in MAM formation (HYOU1, GRP75/HSPA9, GRP78/HSPA5) ( Fig. 7g-i ), autophagy regulation (RUBCN, MTOR) ( Supplementary Fig. 7a-b ), and lipid metabolism (CD36) ( Fig. 7j ) were more prominently expressed in AC compared to PA tissues, especially in monocytes and dendritic cells. FKBP2, a gene highly correlated with HYOU1 in the Human Protein Atlas scRNA-seq dataset (r = 1.000), was similarly enriched in the AC condition ( Supplementary Fig. 7c ), suggesting its involvement in the HYOU1-associated regulatory network. To quantify expression differences between disease and control, we performed pseudobulk analysis. This approach confirmed increased expression of HYOU1, GRP75, GRP78, FKBP2, CD36, RUBCN, and MTOR in AC tissues, particularly in monocytes and dendritic cells ( Fig. 7k-l ). These data indicate a disease-associated upregulation of genes linked to MAM integrity, autophagy, and lipid handling. Together, these single-cell transcriptomic analyses reinforce the pathological relevance of the HYOU1-dependent gene network in atherosclerotic immune cell populations and support its role in autophagy suppression and metabolic dysfunction in vascular inflammation. We investigated the potential association between HYOU1 and disease progression by analyzing a proteomics dataset of monocytes from atherosclerosis patients (PXD041367) in the PRIDE database (https://www.ebi.ac.uk/pride/). 36 Our analysis revealed that HYOU1 expression was upregulated in patients compared to normal controls and increased as the disease progressed ( Fig. 7m ). Notably, HYOU1 levels positively correlated with CD36 ( Fig. 7n ), which was also upregulated in the disease group in single-cell RNA sequencing analysis ( Fig. 7j ). Furthermore, proteomics analysis revealed a positive correlation between HYOU1 and GRP78 protein levels ( Fig. 7o ), consistent with findings from single-cell RNA sequencing analysis ( Fig. 7i ). These results suggest that these ER chaperones may be co-regulated in the context of atherosclerosis, highlighting the potential role of ER stress in disease progression through the coordinated regulation of HYOU1 and GRP78. In summary, our results identify HYOU1 as a novel regulator of the IP 3 R-GRP75-VDAC1 complex in the MAM, which plays a critical role in controlling autophagy and mitigating autophagy-related disease phenotypes, particularly atherosclerosis ( Fig. 8 ). Targeting HYOU1 may therefore represent a promising therapeutic strategy for treating related diseases. Discussion Here, we uncover a novel link between HYOU1, MAM regulation, autophagy, and atherosclerosis. Although organelle interactions are increasingly recognized as key regulators of cellular homeostasis, few studies have explored their therapeutic modulation in human disease. Our findings demonstrate that cryptotanshinone (CTS), a natural anti-atherosclerotic compound, directly targets HYOU1 to rewire ER-mitochondria communication and enhance autophagy. This work positions HYOU1 as a critical regulator of MAM stability and highlights CTS as a promising therapeutic agent for metabolic disorders (Fig. 7 p). Using a label-free target identification approach, we identified HYOU1 as the biological target of CTS. CTS binds the nucleotide-binding domain (NBD) of HYOU1, disrupting its interaction with GRP78 and IP 3 R, leading to the dissociation of the IP 3 R-GRP75-VDAC1 complex at the MAM. This reduces excessive calcium transfer from the ER to mitochondria, alleviating mitochondrial overload and ATP depletion. Mitochondrial calcium overload is a known driver of mROS production, which further exacerbates ER stress, lipid peroxidation, and inflammatory signaling. 37 – 41 Autophagy dysfunction amplifies this oxidative stress, as impaired clearance of damaged mitochondria leads to sustained mROS accumulation (Fig. 7 p, upper panel). This creates a vicious cycle where mROS further suppresses autophagy and promotes atherosclerosis progression. Our findings suggest that CTS indirectly reduces mROS levels by restoring autophagy, rather than acting as a direct antioxidant. By preserving mitochondrial quality control mechanisms, CTS prevents excessive mROS production and mitigates oxidative damage. Molecular modeling suggests that CTS interacts with R217 and N410 in HYOU1’s NBD. Mutagenesis studies confirmed that these residues are critical for CTS binding and autophagy activation, as CTS failed to protect mutant HYOU1 from proteolysis and did not effectively induce autophagosome formation (Fig. 5 c-d). Recent studies indicate that ATP binding to HYOU1 induces a conformational change that enhances its interaction with GRP78, with the N410 residue near the ATP binding site playing a critical role in this interaction. 19 , 42 CTS binds to N410, allosterically modulating ATP binding and destabilizing the HYOU1-GRP78 complex, potentially influencing its interaction with IP 3 R. These findings establish HYOU1 as a key regulator of ER-mitochondria crosstalk, autophagy, and oxidative stress balance, further supporting its role in the atherosclerotic cycle (Fig. 7 p). As illustrated in Fig. 7 p (lower panel), our study reveals that HYOU1-dependent MAM regulation plays a key role in the atherosclerosis. The continuous loop of oxLDL exposure, ER stress, excessive mitochondrial calcium influx, ROS accumulation, and autophagy impairment drives atherosclerosis progression. Our findings suggest that CTS breaks this pathological cycle by restoring MAM homeostasis, reducing calcium overload, and enhancing autophagy, ultimately mitigating atherosclerosis development. Although our experimental models were limited to in vitro and murine systems, we analyzed publicly available single-cell RNA sequencing data from human atherosclerotic plaques to assess the clinical relevance of our findings. The observed upregulation of HYOU1 and its associated regulators—GRP75, GRP78, FKBP2, RUBCN, MTOR, and CD36—was most prominent in monocytes, where both differential expression between diseased and normal tissues and co-expression with HYOU1 were clearly observed. This cell-type-specific enrichment aligns with the established role of monocytes in lipid uptake, inflammatory signaling, and foam cell formation during atherosclerosis. 43 While these observations are correlative and do not establish causality, they are consistent with our mechanistic model and support the translational relevance of the HYOU1-centered MAM-autophagy network in human disease. Beyond atherosclerosis, MAM dysfunction contributes to ferroptosis, lipid metabolism disorders, and cancer. Zhang, Z et al. recently identified a ferroptosis inhibitor that targets the sigma-1 receptor (Sig1R), an ER chaperone, demonstrating that MAM regulation can influence ferroptotic cell death. 44 Inspired by these findings, we investigated whether CTS could similarly modulate ferroptosis via MAM regulation. In RSL3 (ferroptosis inducer)-treated HUVECs, CTS simultaneously promoted cell survival and reduced ER-mitochondria tethering, suggesting a potential protective effect ( Supplementary Fig. 8a-b ). Lipidomics analysis further revealed that CTS decreases polyunsaturated phospholipid content, particularly mitochondrial PE 38:4, PE 40:4, and PS 36:4 suggesting a mechanism for its anti-ferroptotic effects ( Supplementary Fig. 8c-e ). These findings not only strengthen the link between ferroptosis and MAM, but suggest that the discovery of MAM-modulating compounds represents a promising strategy for regulating intracellular homeostasis and stress responses. Given the critical role of inflammation in atherosclerosis, we also investigated CTS's effect on T cell activation. We observed that CTS inhibited the MAM formation induced by TCR stimulation in human CD4 T cells. Notably, this was accompanied by a reduction in the expression of T cell activation markers (such as CD44, CD25). These findings suggest a potential link between MAM regulation and T cell activation in the context of the inflammatory response. While the exact mechanism remains to be further explored, these results imply that CTS may modulate MAM dynamics to influence T cell activation, offering new insights into the regulation of inflammation in atherosclerosis. Numerous studies have shown that HYOU1 is highly expressed in various diseases, particularly in cancer. In epithelial ovarian cancer and lung cancer, HYOU1 drives tumor progression and represents a key molecular target with prognostic significance. 45 , 46 Consistent with these findings, we observed that HYOU1 knockout (KO) cells exhibit a slower proliferation rate compared to wild-type (WT) cells ( Supplementary Fig. 6e ), further supporting its role in cell survival and growth regulation. These observations suggest that HYOU1 may function as a key metabolic regulator across multiple disease contexts. While further studies are needed to determine the molecular pathways linking HYOU1 to tumorigenesis, its expression profile highlights its potential as a biomarker and therapeutic target for cancer and metabolic disorders. As summarized in Fig. 7 p, HYOU1 serves as a central regulator of ER-mitochondrial communication and autophagy, modulating cellular stress responses under conditions resembling oxLDL-induced atherosclerosis. CTS directly interacts with the NBD of HYOU1, disrupting MAM-associated calcium transfer, thereby restoring autophagic homeostasis and alleviating lipid accumulation. These findings position HYOU1 as a promising therapeutic target, with CTS-based interventions offering a novel strategy to break the oxLDL-driven atherosclerotic cycle and regulate organelle crosstalk in disease. Materials and Methods Cell Culture Human umbilical vein endothelial cells (HUVECs, up to passage 9) were purchased from Lifeline Cell Technology (Frederick, MD) and cultured in EBM-2 Basal Medium supplemented with growth factors. HEK293, HepG2, HeLa, and RAW264.7 cells were purchased from the Korean Cell Bank (Seoul, South Korea) and cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics. All cell cultures were maintained at 37°C in a humidified incubator with 5% CO2 and pH 7.4. Cells were tested for mycoplasma contamination every two months. CRISPR-Cas9 Knockout Cell Generation Target sequences for single Single-guide RNAs (sgRNAs) were designed using the Cas-Designer (http://www.rgenome.net/cas-designer/). Each sgRNA spacer oligo was cloned into the pRG2 vector, and plasmid sequences were verified by Sanger sequencing. HEK293T cells were transfected with 375 ng of Cas9 and 125 ng of sgRNA-expressing plasmids using the jetOPTIMUS transfection reagent, following the manufacturer’s protocol. Single cells were isolated by limiting dilution, and knockout clones were validated using next-generation sequencing (NGS) with the Illumina MiniSeq system. The NGS data were analyzed using Cas-Analyzer (http://www.rgenome.net/cas-analyzer/). Ethics approval All animal experiments were conducted in accordance with the ethical guidelines approved by the Institutional Animal Care and Use Committee of Yonsei University (IACUC-A-202208-1519-01). Animal Studies ApoE -/- C57BL/6J male mice were maintained in a specific-pathogen-free facility under a 12-hour light/dark cycle with ad libitum access to food and water. Mice (n = 6) were randomly assigned to experimental groups, with body weight matched across groups at the time of allocation. Mice were fed a high-cholesterol diet (Research Diets Inc., New Brunswick, NJ) starting at 6 weeks of age and continued until the conclusion of the study, with the final sacrifice performed at 18 weeks of age. For intraperitoneal injections, the vehicle solution consisted of 30% polyethylene glycol 300 and 5% Tween 80 in saline. At the experimental endpoint, mice were anesthetized using avertin. PLA (Proximity Ligation Assay) Cells were grown on a chambered cell culture slide (SPL Life Sciences, Pocheon, South Korea) and treated with the drug. PLA experiments were performed using the Duolink In Situ Red Kit or NaveniFlex Cell MR Red kit according to manufacturer instructions. Briefly, cells were fixed with 4% paraformaldehyde for 10 minutes and permeabilized with 0.2% Triton X-100 for 20 minutes at room temperature. The cells were then blocked with blocking solution for 1 hour and incubated with primary antibodies overnight at 4°C. PLA probes were applied and incubated for 1 hour at 37°C. Cells were incubated with ligase for 30 minutes, followed by DNA polymerase incubation for 100 minutes. Samples were mounted with mounting medium containing DAPI. PLA signals were observed using an LSM 980 confocal microscope. Quantification of PLA signals was performed by counting dots or measuring fluorescence intensity per cell area. Calcium Analysis To monitor mitochondrial or cytosolic calcium influx, HUVECs were seeded onto chambered coverslips (ibidi, Gräfelfing, Germany, #80827) and incubated with either Rhod-2 AM or Fluo-4 AM in Krebs-Ringer-HEPES (KRH) buffer (pH 7.4) for 30 minutes at 37°C incubator. After washing with KRH buffer, the cells were incubated in calcium-free KRH buffer (pH 7.4) for an additional 30 minutes to allow for the de-esterification of the dyes. Calcium influx was assessed by capturing live-cell images using an LSM980 confocal microscope. Fluorescence intensity within selected regions of interest (ROIs) was quantified over time using ImageJ software. Transmission Electron Microscopy (TEM) HUVECs, HEK293T control cells, and HEK293T HYOU1 knockout (KO) cells were cultured in culture dishes (NEST Biotechnology, Wuxi, China) until they reached 50–60% confluence. Cells were treated with 5 µM CTS or dimethyl sulfoxide (DMSO) as a vehicle control for 24 hours at 36°C. After treatment, cells were fixed in a solution containing 1% glutaraldehyde and 1% paraformaldehyde in 0.3 M cacodylate buffer for 1 hour at 4°C. Fixed samples were post-fixed in 1% osmium tetroxide in distilled water for 1 hour at 4°C. The specimens were then stained with 1% uranyl acetate overnight at 4°C, dehydrated through a graded ethanol series, and embedded in epoxy resin (EMS, Hatfield, PA). Ultrathin sections (~60 nm) were prepared using an ultramicrotome (Leica Microsystems, Wetzlar, Germany) and mounted on copper grids. Sections were stained with UranyLess for 2 minutes, rinsed with distilled water, and counterstained with lead citrate for 1 minute. Transmission electron microscopy (TEM) images were acquired using a Tecnai 20 transmission electron microscope (Thermo Fisher Scientific, Waltham, MA) operating at 120 kV, equipped with a US1000XP camera. Holotomography HUVECs were seeded onto HT-Ready 96 well plate (Tomocube, Daejeon, Republic of Korea). The cells were then treated with either oxLDL alone or a combination of oxLDL and CTS for 24 hours. High-resolution live cell images were acquired using HT-X1 holotomographic microscope (Tomocube), which utilizes low-coherence light sources. During imaging, the plate was maintained in a wet chamber with controlled temperature (37°C) and CO2 levels (5%). Images were analyzed using AI-based TomoAnalytic 2.0 software (Tomocube). Lipidomics HUVECs treated with or without 5 µM CTS were harvested, and mitochondria were isolated using a mitochondria isolation kit according to the manufacturer’s instructions. A portion of the mitochondrial pellet was resuspended in 2% CHAPS in Tris-buffered saline (pH 7.2), and protein concentration was determined using a BCA assay. The remaining pellet was stored at -80°C for subsequent lipidomic analysis. Detailed methods are provided in the supplementary material. DARTS-LC-MS/MS Analysis Membrane proteins were extracted from HepG2 and HUVEC cells using a Membrane Protein Extraction Kit, and protein concentration was adjusted to 1 mg/mL. All steps were performed on ice to minimize protein degradation. Protein samples were incubated with either DMSO or CTS at 4°C for 3 hours with gentle rocking. After incubation, pronase was added to each sample, and digestion was carried out at room temperature for 10 minutes. The reaction was stopped by adding 6× SDS sample buffer, followed by heat denaturation. The digested protein samples were separated by SDS-PAGE, and in-gel digestion was performed. Peptides were analyzed using a Triple TOF 6600 mass spectrometer (AB Sciex, Concord, Ontario, Canada) coupled with an Acquity UPLC system (Waters, Milford, MA). Both information-dependent acquisition (IDA) and sequential window acquisition of all theoretical fragment-ion spectra (SWATH) tandem mass spectrometry methods were applied. CETSA analysis HEK293 cells were harvested using trypsin-EDTA solution. Cell suspensions at a concentration of 3×107 cells/15 mL was aliquoted into each conical tube, CTS was added, with DMSO used as a control. The cells were incubated for 1 hour in a CO2 incubator at 37 °C with gently mixing. After centrifugation, the pellet was washed with PBS and resuspended in 1 mL of PBS (containing protease inhibitors). The suspension was aliquoted into PCR tubes (~100 μL per tube) and heated between 40-64°C for 3 minutes, followed by cooling to 25°C for an additional 3 minutes in a thermal cycler. The tubes were then centrifuged to pellet the cells and the supernatant was discarded. The pellet was resuspended in 0.4% NP-40 (in PBS) supplemented with protease inhibitors to facilitate the dissolution of hydrophobic proteins. The cell suspension was subjected to two cycles of freezing-thawing in liquid nitrogen and centrifuged at 20,000 g for 20 min at 4 °C. The supernatants (soluble proteins) were collected and used for western blotting. Microscale thermophoresis Fluorescence labeling of purified HYOU1 protein was performed using the Protein Labeling Kit RED-NHS (NanoTemper Technologies, Munich, Germany), according to the manufacturer’s protocol. Binding assays were performed using the Monolith NT.115pico instrument (NanoTemper Technologies). Labeled protein was prepared at a final concentration of 20 nM in assay buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl) supplemented with 0.05% Tween-80 to minimize protein aggregation. The labeled protein was mixed with serially diluted ligand samples and loaded into premium capillaries (NanoTemper Technologies). Thermophoresis was measured and the dissociation constant (Kd) was determined. Data analysis was performed using MO Affinity Analysis software (NanoTemper Technologies). In silico docking study Molecular docking analysis was performed using Discovery Studio 2018 software (BIOVIA, San Diego, CA). The 3D structure of HYOU1 was obtained from the Alphafold database, and the ligand structure was obtained from the Pubchem. For the docking simulations, the CDOCKER method, a grid-based docking technique, was employed to predict the binding mode of the ligands to the target protein. The Binding sites on protein were defined based on receptor’s cavities, then the ligand was docked into these binding sites. The top 10 docking poses were generated based on the binding affinity, and the binding energy (CDOCKER energy) for each pose was calculated. scRNA-seq data analysis Single-cell RNA sequencing (scRNA-seq) data were analyzed using Seurat (version 5) in R Studio(V 4.4.1). 47, 48 Seurat objects were created for each sample, with PA tissues labeled as Normal and AC plaques as Disease. After merging, QC filtering retained cells with >200 but <6,000 detected genes and <5% mitochondrial reads. The data were normalized using the SCTransform method, 49 and highly variable genes were identified. Principal component analysis (PCA) was used for dimensionality reduction, and the top components were used for shared nearest neighbor (SNN)-based clustering (resolution = 0.8) and UMAP visualization.. Harmony was applied for batch correction. Cell type annotation was based on marker genes identified using FindAllMarkers (min.pct = 0.25, logFC > 0.25) and assigned to known cell types, including T cells, monocytes, dendritic cells, endothelial cells, smooth muscle cells, fibroblasts, plasma cells, mast cells, and natural killer cells. 35, 50 Pseudobulk analysis was performed to aggregate scRNA-seq data at the sample level for bulk-like differential expression analysis using Seurat and DESeq2. 47 Gene expression counts were summed by sample ID and cell type, and raw counts were normalized using variance-stabilizing transformation (VST) in DESeq2. Statistics Six mice per group were used in this study. Sample sizes were determined based on institutional guidelines, with a minimum of five mice per group to ensure consistent and reliable results across the experimental groups. No statistical method was used to predetermine the sample size. Since the researchers had to identify the cages of the mice for treatments, blinding could not be applied. All the data were analyzed using Graph Pad Prism 9.0 (GraphPad Software Inc., San Diego, CA) and expressed as mean ± SD. Comparison between two groups was done by the unpaired Students’ t-test. Minimum of three independent experiments were conducted to allow for statistical comparisons. P-values less than 0.05 were considered statistically significant (* p < 0.05, ** p < 0.01, *** p < 0.001, ****p<0.0001). Declarations Data Availability Single-cell transcriptome profiles of human calcified atherosclerotic core (AC) plaques and patient-matched proximal adjacent (PA) portions of carotid artery tissue were obtained from the Gene Expression Omnibus (GEO) database (GSE159677, PMID: 36224302, DOI: 10.1038/s42003-022-04056-7). 35, 50 Acknowledgments We would like to thank Dongjin Lee (Yonsei University, Republic of Korea) for preparing DARTS samples for MS analysis. We also express our gratitude to Dr. Dong-Hyung Cho (Kyungpook National University, Republic of Korea) for providing the SPLICS vector, Dr. Hae-Ryung Park (University of Rochester, USA) for supplying the CHOP-mCherry reporter cells, and Dr. Seong-Woon Yu (DIGIST, Republic of Korea) for supplying the ATG3 knockout HeLa cells. We are also grateful to Dr. Goo Taeg Oh (Ewha Womans University, Republic of Korea) for generously gifting us ApoE-/- mice. We also thank Dr. Sumin Lee and Dr. Hyejin Kim (Tomocube, Inc., Republic of Korea) for their assistance with holotomography imaging and AI-based analysis. We also thank Dr. Eun-woo Lee (KRIBB, Republic of Korea) for providing the reagent (RSL3) and offering advice for related experiments. This work was partly supported by grants from the National Research Foundation of Korea, funded by the government of the Republic of Korea: 2021R1A3B1077371 (H.J.K.), 2022R1A2C1009376 (J.Y.M.), 2021R1C1C1012076 (J.Y.), 2021M3E5E3080529 (J.Y.), 2022R1A2C2093050 (M.L.), 2021M3A9H3015389 (S.B.), 2022M3H9A2096186 (J.Y.K.), 2021R1A2C2003171 (M.H.M.) and the Brain Korea 21 Plus Project (H.J.K.). This work was also supported by the ICONS (Institute of Convergence Science, Yonsei University) (H.J.K.), the Research Program of the National Research Council of Science & Technology CRC22021-100 (J.Y.K), and the Korea Basic Science Institute Research Grant No. A423200 (J.Y.K.). Author Contributions M.K. and H.J.K. planned and conceptualized the study. M.K. performed most experiments with contributions from J.H., S.K., H.E.L., J.Y.M., D.Y., J.Y., H.C., M.L., Y.L., S.B., S.S., J.Y.L., J.Y.K., and M.H.M. M.K. and H.J.K. analyzed data, performed statistics, and wrote the manuscript. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6301868","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":433632860,"identity":"49bf84db-22c6-494c-90b6-e777880ee1d1","order_by":0,"name":"Minjeong Ko","email":"","orcid":"https://orcid.org/0000-0003-1952-1036","institution":"Yonsei University","correspondingAuthor":false,"prefix":"","firstName":"Minjeong","middleName":"","lastName":"Ko","suffix":""},{"id":433633364,"identity":"e20a11ed-7c95-4f3c-9531-1dee9ed746b8","order_by":1,"name":"Jungmin 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b\u003c/strong\u003e Foam cell formation in RAW264.7 macrophages treated with 50 µg/mL oxLDL and 5 µM Tan II A, DHT, or CTS for 48 hours. Scale bar, 20 µm. \u003cstrong\u003ec \u003c/strong\u003eTEM images of HUVECs treated with 50 µg/mL oxLDL for 20 hours, with or without subsequent 4-hour CTS treatment. Arrows indicate ER-mitochondria contact sites. \u003cstrong\u003ed\u003c/strong\u003e Proximity ligation assay (PLA) detecting ER-mitochondria interactions in cells treated as in (\u003cstrong\u003ec\u003c/strong\u003e). \u003cstrong\u003ee\u003c/strong\u003e Real-time mitochondrial calcium levels measured by confocal microscopy in cells loaded with 2 µM Rhod-2, a mitochondrial Ca²⁺ indicator. Mitochondrial calcium fluorescence was recorded at baseline and after ER Ca²⁺ release induced by 200 µM histamine.\u003cstrong\u003e f\u003c/strong\u003e Relative ATP levels measured after 5 μM CTS treatment. \u003cstrong\u003eg\u003c/strong\u003e Phospholipid composition of mitochondria isolated from HUVECs treated with 5 µM CTS, analyzed by mass spectrometry. PI, phosphatidylinositol; PS, phosphatidylserine; PE, phosphatidylethanolamine; PEp, ether PE; PC, phosphatidylcholine; PCp, ether PC. \u003cstrong\u003eh\u003c/strong\u003e Representative holotomography images of HUVECs treated with 5 µM CTS and 50 µg/mL oxLDL for 24 hours. Scale bar, 20 µm. \u003cstrong\u003ei \u003c/strong\u003eAI-based phenotypic clustering of cells following drug treatment. \u003cstrong\u003ej \u003c/strong\u003eRepresentative images showing mCherry fluorescence in mCherry-CHOP stable HEK293 cells co-treated with 50 μg/mL oxLDL and 5 μM CTS for 48 hours. \u003cstrong\u003ek\u003c/strong\u003e Cells were co-treated with 50 μg/mL oxLDL and 5 μM CTS for 24 hours, and mROS levels were measured using 2 μM MitoSOX. Quantitative data are presented as mean ± SD. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6301868/v1/5d81b253d4d89aa8effff293.png"},{"id":79402240,"identity":"c1afb5ab-e038-4266-ae03-304418cfbaef","added_by":"auto","created_at":"2025-03-28 02:56:57","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":495800,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCTS induces autophagy and lipid degradation via calcium signaling.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e Lysotracker and MitoTracker staining of cells co-treated with 50 µg/mL oxLDL and 5 µM CTS for 48 hours. Scale bar, 10 μm. \u003cstrong\u003eb\u003c/strong\u003e Western blot analysis of autophagy markers in HUVECs treated with 5 µM CTS for 24 to 72 hours. \u003cstrong\u003ec\u003c/strong\u003e Western blot analysis of LC3-II levels in HUVECs treated with 5 µM CTS, with or without 20 μM chloroquine (CQ). Co-treatment with CTS and CQ for 24 hours increased LC3-II accumulation. \u003cstrong\u003ed\u003c/strong\u003e Cytosolic calcium levels in HUVECs treated with 5 µM CTS, visualized using the Fluo-4 calcium indicator (1 µM). Scale bar, 10 μm. \u003cstrong\u003ee\u003c/strong\u003eNuclear and cytoplasmic fractions from CTS-treated HUVECs analyzed for TFEB nuclear translocation by immunoblotting. Lamin A/C and GAPDH were used as nuclear and cytoplasmic markers, respectively. \u003cstrong\u003ef\u003c/strong\u003e HUVECs treated with 5 μM CTS and 1 nM bafilomycin A1 (BafA1) for 48 hours, with or without 50 μg/mL DiI-conjugated oxLDL. oxLDL uptake was detected using DiI fluorescence. Scale bar, 20 μm. \u003cstrong\u003eg-h\u003c/strong\u003e Intracellular lipid levels in wild-type and ATG3-knockout HeLa cells treated with 5 µM CTS for 24 hours, assessed by BODIPY staining. Quantitative data are presented as mean ± SD. *p \u0026lt; 0.05, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001, ns = not significant.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6301868/v1/bcaa53b510e0f5c50cc395bc.png"},{"id":79403325,"identity":"7d946abb-fe6e-463b-aa98-a337f0c38f25","added_by":"auto","created_at":"2025-03-28 03:12:57","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":617965,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCTS alleviates atherosclerosis in ApoE-/- mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea \u003c/strong\u003eApoE⁻/⁻ mice were fed a high-cholesterol diet starting at 6 weeks of age to induce atherosclerosis. From 10 weeks of age, vehicle or CTS (20 mg/kg) was administered intraperitoneally every 2 days for 8 weeks (n=6). \u003cstrong\u003eb\u003c/strong\u003e Body weights of mice measured every 4 days. \u003cstrong\u003ec-d\u003c/strong\u003e. Serum levels of total cholesterol (T. Chol) and triglycerides (TG) were reduced in CTS-treated mice. \u003cstrong\u003ee \u003c/strong\u003eOil Red O staining of aortic sinus cross-sections. Lesion areas were quantified using ImageJ. Scale bar, 200 µm. \u003cstrong\u003ef \u003c/strong\u003eEn face Oil Red O staining of whole aortas to visualize plaques. Lesion areas were quantified as a percentage of the total area. Scale bar, 5 mm. \u003cstrong\u003eg\u003c/strong\u003e Immunofluorescence staining of LC3B (autophagosome) and BODIPY (lipids) in primary peritoneal macrophages isolated from each group of mice. Scale bar, 10 µm. \u003cstrong\u003eh\u003c/strong\u003e Proximity ligation assay (PLA) detecting IP\u003csub\u003e3\u003c/sub\u003eR-VDAC1 interactions in paraffin-embedded aortic sinus tissue. Scale bar, 10 µm. \u003cstrong\u003ei\u003c/strong\u003e PLA analysis of IP\u003csub\u003e3\u003c/sub\u003eR-VDAC1 interactions in primary peritoneal macrophages. Scale bar, 10 µm. Quantitative data are presented as mean ± SD. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6301868/v1/414549492192b70b8582510f.png"},{"id":79403327,"identity":"15580736-b341-4637-960a-7c8e822c4f32","added_by":"auto","created_at":"2025-03-28 03:12:57","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":572435,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDARTS-LC-MS/MS analysis identifies HYOU1 as a potential target of CTS.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e Schematic representation of the DARTS-LC-MS/MS workflow. Membrane proteins from HepG2 cell lysates were incubated with either DMSO or CTS for 3 hours, followed by pronase digestion under three conditions: M1 (control), M2 (DMSO + pronase), and M3 (CTS + pronase). Samples were processed via in-gel digestion, and SWATH-MS analysis was performed in triplicate on the same set of samples to ensure reproducibility. Principal component analysis (PCA) revealed distinct clustering between groups. \u003cstrong\u003eb \u003c/strong\u003eTarget candidate protein selection based on DARTS-LC-MS/MS results. \u003cstrong\u003ec \u003c/strong\u003eSubcellular distribution of CTS target candidates. Proteins with the highest sequence coverage were predominantly localized in the ER and mitochondria. \u003cstrong\u003ed\u003c/strong\u003e Functional network analysis of CTS target candidates using STRING database. \u003cstrong\u003ee\u003c/strong\u003e Heatmap showing pronase resistance patterns of ER-localized candidate proteins upon CTS binding. \u003cstrong\u003ef\u003c/strong\u003e DARTS assay in HUVECs incubated with CTS at increasing concentrations for 3 hours at 4°C, followed by 1 µg/mL pronase treatment for 10 minutes at RT. HYOU1 band intensity was quantified and plotted as a sigmoid curve to estimate CTS binding affinity. \u003cstrong\u003eg \u003c/strong\u003eCETSA experiment was performed in live HEK293 cells. Cells were incubated with CTS at 37°C for 1 hour, followed by heat treatment between 40-64°C. The resistance of HYOU1 to thermal denaturation was then assessed. \u003cstrong\u003eh\u003c/strong\u003eMicroscale thermophoresis (MST) analysis showing the binding of CTS to HYOU1 with a dissociation constant (Kd) of 1.42 μM.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6301868/v1/a2f3efea6b4be60ab9181621.png"},{"id":79402253,"identity":"910af4f2-fdbc-4a90-89ec-db0bd071cf41","added_by":"auto","created_at":"2025-03-28 02:56:57","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":622665,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of critical residues in the CTS-HYOU1 interaction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea \u003c/strong\u003eIn silico molecular docking analysis of CTS binding to the predicted 3D structure of HYOU1 (AF-Q9Y4L1-F1).\u003cstrong\u003e b\u003c/strong\u003e Predicted binding site residues of the HYOU1-CTS complex. \u003cstrong\u003ec\u003c/strong\u003e DARTS assay was performed using membrane protein fractions from HEK293 cells transfected with MYC-tagged HYOU1 wild-type (WT), R217A, or N410D mutants for 48 hours. Ectopic expression of HYOU1 was confirmed by anti-MYC immunoblotting. \u003cstrong\u003ed\u003c/strong\u003e LC3B immunofluorescence staining of autophagosomes in HUVECs transfected with HYOU1 wild-type (WT), R217A, or N410D mutants, and treated with 5 µM CTS. Transfection efficiency was confirmed by MYC staining.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6301868/v1/d65b79b66021a1fad3d14ea9.png"},{"id":79402428,"identity":"92daf0ff-3813-412f-9049-458bc016214b","added_by":"auto","created_at":"2025-03-28 03:04:57","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":533896,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCTS reduces ER-mitochondria contacts by targeting HYOU1, highlighting its role in atherosclerosis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea \u003c/strong\u003ePLA analysis showing the interactions between proteins involved in MAM formation.\u003cstrong\u003e b\u003c/strong\u003e Immunoprecipitation of IP\u003csub\u003e3\u003c/sub\u003eR from HUVEC lysates with or without CTS, followed by Western blotting to assess interacting partners. \u003cstrong\u003ec\u003c/strong\u003e Schematic of the MAM complex and its regulatory components. \u003cstrong\u003ed\u003c/strong\u003e PLA assay to evaluate the effect of HYOU1 knockdown on the interactions between ER(IP\u003csub\u003e3\u003c/sub\u003eR) and mitochondria (VDAC1). Scale bar: 10 µm. \u003cstrong\u003ee\u003c/strong\u003e Representative images of cytosol calcium levels assessed using Fluo-4 reagent by confocal microscopy after HYOU1 knockdown. Scale bar: 20 µm. \u003cstrong\u003ef\u003c/strong\u003e Immunoblotting to assess the levels of HYOU1, SQSTM1, and LC3 after transfection with si HYOU1 (20 nM) for 48 hours. \u003cstrong\u003eg\u003c/strong\u003e Representative images of lipid staining with BODIPY in HYOU1-silenced HUVECs treated with oxLDL. Scale bar: 20 µm. \u003cstrong\u003eh\u003c/strong\u003e RAW264.7 cells were transfected with 100 nM mouse Hyou1 siRNA and incubated with 50 µg/mL oxLDL for 48 hours. Scale bar: 20 µm.\u0026nbsp; Foam cell formation was evaluated by Oil Red O staining. Quantitative data are presented as mean ± SD. **p \u0026lt; 0.01, ***p\u0026lt;0.001, ****p \u0026lt; 0.0001, ns = not significant.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6301868/v1/26fad53e935f327d9e6aa2a3.png"},{"id":79402249,"identity":"d2f70212-3ff7-4fad-8c85-2eba54f90e29","added_by":"auto","created_at":"2025-03-28 02:56:57","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1437694,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHYOU1 links ER-mitochondria communication and autophagy in atherosclerosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e Western blot analysis confirming HYOU1 expression levels in CRISPR-Cas9-generated HYOU1 heterozygous knockout (KO) cells. \u003cstrong\u003eb \u003c/strong\u003eRepresentative images of PLA analysis showing interactions between the ER and mitochondria (IP\u003csub\u003e3\u003c/sub\u003eR-VDAC1) in wild-type (WT) and HYOU1 knockout (KO) 293T cells. Scale bar: 10 µm.\u003cstrong\u003e c\u003c/strong\u003e Representative TEM images of HYOU1 KO cells treated with DMSO or 5 µM for 4 hours. Green, mitochondria; red, ER; arrows, ER-mitochondria contact sites. \u003cstrong\u003ed\u003c/strong\u003e PLA analysis of IP\u003csub\u003e3\u003c/sub\u003eR-VDAC1 interactions in HYOU1 KO cells 4 hours after CTS treatment. \u003cstrong\u003ee \u003c/strong\u003eBODIPY staining in HYOU1 KO cells 24 hours after CTS treatment. \u003cstrong\u003ef \u003c/strong\u003eUMAP visualization of cell type annotation in single-cell RNA-seq analysis from human calcified atherosclerotic core (AC-Disease, n=3) and patient-matched proximal adjacent (PA-Control, n=3) tissues.\u003cstrong\u003e g-j \u003c/strong\u003eFeaturePlot visualization of the gene expression patterns of selected genes (HYOU1, HSPA9 (GRP75), HSPA5 (GRP78), and CD36) in AC-Disease and PA-Control tissues. \u003cstrong\u003ek\u003c/strong\u003e Dot plot visualization of gene expression in AC-Disease and PA-Control using Pseudobulk analysis. \u003cstrong\u003el \u003c/strong\u003eThis heatmap illustrates the pseudobulk gene expression patterns of selected genes across various cell types in AC-Disease and PA-Control. \u003cstrong\u003em \u003c/strong\u003eHYOU1 protein levels in monocytes from healthy individuals (n = 17) and patients with early (n = 18) or advanced (n = 16) atherosclerosis. \u003cstrong\u003en-o \u003c/strong\u003ePearson correlation analysis of HYOU1 expression with CD36, and GRP78 protein levels in monocytes from atherosclerosis patients (n = 34). \u003cstrong\u003ep\u003c/strong\u003e ① Targeting HYOU1 with CTS disrupts the IP\u003csub\u003e3\u003c/sub\u003eR-GRP75-VDAC1 complex, ② resulting in reduced ER-mitochondria interactions. ③ This increases cytosolic calcium levels and promotes nuclear translocation of TFEB while decreasing mitochondrial calcium and ATP levels. ④ As a result, autophagy is induced, ⑤ reducing oxLDL levels ⑥ along with alleviating cellular stress, including ER stress and mROS levels. Quantitative data are presented as mean ± SD. *p \u0026lt; 0.05, ns = not significant.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6301868/v1/60804fa3b50cdb47b19338aa.png"},{"id":79404215,"identity":"8ce49d8d-f03b-4e06-a3ba-abf772222a56","added_by":"auto","created_at":"2025-03-28 03:29:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6853335,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6301868/v1/c59ae1be-649a-4f11-a6d3-ca7f636a2cd1.pdf"},{"id":79402241,"identity":"0f3fb887-33c9-4f07-955b-f4307fe9e251","added_by":"auto","created_at":"2025-03-28 02:56:57","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":341856,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical Abstarct\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6301868/v1/e595ba5816fd0eb221a74d41.png"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eCryptotanshinone Targets HYOU1 to Rewire ER-Mitochondria Communication and Enhance Autophagy in Atherosclerosis\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCardiovascular disease (CVD) remains the leading cause of mortality worldwide, with atherosclerosis being a primary contributor. Atherosclerosis is driven by lipid accumulation, chronic inflammation, and endothelial dysfunction, ultimately leading to plaque formation and vascular complications.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e Oxidized low-density lipoprotein (oxLDL), a key driver of atherosclerosis, induces oxidative stress, endothelial damage, and macrophage activation, accelerating plaque formation.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e Lipid accumulation in atherosclerosis triggers cellular stress including endoplasmic reticulum (ER) stress, disrupting signaling pathways that drive disease progression.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e Beyond these well-known effects, oxLDL also perturbs intracellular homeostasis by enhancing the formation of mitochondria-associated ER membranes (MAMs), leading to excessive mitochondrial calcium influx and impaired autophagy.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e Given that autophagy is crucial for lipid clearance and inflammation resolution, its dysregulation, combined with increased mitochondrial reactive oxygen species (mROS) production, establishes a pathological loop that accelerates atherosclerosis progression. We define this feedback mechanism as the 'oxLDL-driven atherosclerotic cycle,' where oxLDL-induced MAM formation suppresses autophagy and promoting excessive mROS generation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Elevated mROS further drive oxidation of native LDL, leading to increased oxLDL accumulation, thereby reinforcing the cycle.\u003c/p\u003e \u003cp\u003eOrganelle crosstalk plays a fundamental role in maintaining cellular homeostasis, with MAM serving as a critical interface for ER- mitochondria interactions.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e The MAM regulates key processes such as calcium transport, lipid metabolism, mitochondrial dynamics, and autophagy.\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e One of the major protein complexes, the IP\u003csub\u003e3\u003c/sub\u003eR-Grp75-VDAC1 complex, facilitates calcium flux between the ER and mitochondria.\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e Loss of MAM integrity due to impaired VAPB (ER protein) and PTPIP51 (mitochondrial protein) disrupt calcium exchange and subsequently induces autophagy.\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e Furthermore, mitofusin-2 (MFN2), a mitochondrial fusion protein, links the ER and mitochondria, interacting with AMPK to regulate autophagy under metabolic stress.\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e Despite growing evidence that MAM integrity is essential for maintaining metabolic balance, therapeutic strategies targeting MAM in atherosclerosis remain largely unexplored. While MAM dysfunction has been extensively studied in neurodegenerative and metabolic disorders, its role in cardiovascular diseases, particularly atherosclerosis, remains poorly understood.\u003c/p\u003e \u003cp\u003eNatural compounds have gained attention for their potential to modulate key signaling pathways, including those governing metabolism and organelle dynamics.\u003csup\u003e\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e Cryptotanshinone (CTS), a bioactive component of Danshen (\u003cem\u003eSalvia miltiorrhiza\u003c/em\u003e), has demonstrated anti-atherosclerotic effects.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e However, its impact on MAM dynamics and molecular targets remains unexplored. Identifying the cellular targets of CTS could provide new insights into regulatory pathways governing organelle crosstalk and autophagy.\u003c/p\u003e \u003cp\u003eIn this study, we investigate the potential of Hypoxia Up-regulated Protein 1 (HYOU1, also known as GRP170) as a target for CTS-mediated modulation of ER-mitochondrial interactions. HYOU1 is an ER-resident chaperone involved in protein quality control and the regulation of the unfolded protein response (UPR), which is activated by ER stress.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e HYOU1 contains a nucleotide-binding domain (NBD) that mediates its chaperone activity through ATP/ADP binding.\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e Although HYOU1\u0026rsquo;s role in atherosclerosis has been underexplored, recent studies have shown that targeting its NBD can suppress pathogenic fibroblast activity.\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e Given that HYOU1 may regulate MAM integrity and metabolic stress, targeting its NBD could represent a therapeutic approach for modulating ER-mitochondrial crosstalk in atherosclerosis.\u003c/p\u003e \u003cp\u003eHere, we explore the role of MAM integrity and autophagy in atherosclerosis and examine whether targeting this pathway could provide a therapeutic strategy. We assess the effects of pharmacological modulation of MAM and investigate the contribution of HYOU1 to metabolic stress in atherosclerosis. These findings may offer new insights into the molecular mechanisms linking ER-mitochondrial crosstalk, autophagy, and lipid accumulation, providing a potential framework for therapeutic strategies targeting organelle communication in cardiovascular disease.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eCTS Regulates ER-Mitochondria Interactions to Mitigate oxLDL-Induced Cellular Dysfunction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe define the \u0026lsquo;ox-LDL driven atherosclerotic cycle\u0026rsquo; as the feedback loop initiated by oxLDL, which triggers the formation of MAMs, suppresses autophagy, and induces cellular stress (\u003cstrong\u003eFig. 1a\u003c/strong\u003e). This cycle exacerbates atherosclerosis progression by disrupting cellular homeostasis. To explore the impact of oxLDL on cellular physiology, we treated HUVECs with 50 \u0026mu;g/mL oxLDL, which inhibited cell proliferation at both 24 and 48 hours (\u003cstrong\u003eSupplementary Fig. 1a\u003c/strong\u003e). We further confirmed that oxLDL enhances ER-mitochondrial interactions, demonstrated by GFP fluorescence complementation using the SPLICS system (\u003cstrong\u003eSupplementary Fig. 1b\u003c/strong\u003e),\u003csup\u003e21\u003c/sup\u003e and proximity ligation assay (PLA), a technique that detects protein-protein interactions within close proximity, applied to IP\u003csub\u003e3\u003c/sub\u003eR-VDAC1 pairs (\u003cstrong\u003eSupplementary Fig. 1c\u003c/strong\u003e). This interaction led to elevated mitochondrial calcium levels, a response that could be blocked by the IP\u003csub\u003e3\u003c/sub\u003eR antagonist xestospongin C, highlighting a role for IP\u003csub\u003e3\u003c/sub\u003eR in ER-mitochondrial calcium transport (\u003cstrong\u003eSupplementary Fig. 1d\u003c/strong\u003e). Additionally, oxLDL induced modest ER-lysosome interactions but did not significantly affect mitochondria-lysosome interactions (\u003cstrong\u003eSupplementary Fig. 1e-f\u003c/strong\u003e). Importantly, oxLDL treatment impaired autophagic flux, as evidenced by a decrease in TFEB nuclear translocation and the accumulation of SQSTM1, LC3-II, and autophagosomes (\u003cstrong\u003eSupplementary Fig. 1g-i\u003c/strong\u003e). These impairments were associated with increased expression of CHOP, a marker of ER stress (\u003cstrong\u003eSupplementary Fig. 1j-k\u003c/strong\u003e).\u003csup\u003e22\u003c/sup\u003e oxLDL treatment also disrupted mitochondrial function, leading to elevated mitochondrial ROS (mROS) levels, swelling, and fragmentation (\u003cstrong\u003eSupplementary Fig. 1l-m\u003c/strong\u003e). These findings highlight the role of the atherosclerotic cycle in exacerbating cellular dysfunction driven by oxLDL.\u003c/p\u003e\n\u003cp\u003eWe explore potential therapeutic approaches aimed at disrupting this cycle to mitigate the pathological effects associated with atherosclerosis. Our previous study indicated that a \u003cem\u003eSalvia miltiorrhiza\u003c/em\u003e (Danshen) extract reduced atherosclerosis via autophagy.\u003csup\u003e17\u003c/sup\u003e We investigated the active compounds of Danshen for their ability to inhibit foam cell formation, a hallmark of atherosclerosis, in oxLDL-stimulated RAW264.7 macrophages. Among the major lipophilic compounds, cryptotanshinone (CTS) exhibited superior inhibitory activity on foam cell formation compared to tanshinone-II-A (Tan II A) and dihydrotanshinone-I (DHT) (\u003cstrong\u003eFig. 1b\u003c/strong\u003e). Next, we examined the effects of CTS on oxLDL-induced MAM dynamics in HUVECs. An MTT assay revealed that the IC\u003csub\u003e50\u003c/sub\u003e of CTS was approximately 5 \u0026micro;M in HUVECs (\u003cstrong\u003eSupplementary Fig. 2a\u003c/strong\u003e). Using transmission electron microscopy (TEM), we observed that 4-hour CTS treatment decreased ER-mitochondria contact points within 40 nm, from 61% to 37%, in oxLDL-treated HUVECs, without altering mitochondrial area or circularity (\u003cstrong\u003eFig. 1c\u003c/strong\u003e, \u003cstrong\u003eSupplementary Fig. 2b-d\u003c/strong\u003e). CTS treatment reduced mitochondrial swelling and the oxLDL-induced increase in ER-mitochondria contact area (\u003cstrong\u003eFig. 1c\u003c/strong\u003e). PLA experiments revealed that oxLDL treatment increased IP\u003csub\u003e3\u003c/sub\u003eR-VDAC1 interactions, which were reduced upon CTS treatment (\u003cstrong\u003eFig. 1d\u003c/strong\u003e). Importantly, CTS did not affect the expression levels of IP\u003csub\u003e3\u003c/sub\u003eR or VDAC1 within the 4 hours of treatment (\u003cstrong\u003eSupplementary Fig. 2e\u003c/strong\u003e). To assess microtubule involvement, cells were treated with tubulin polymerization inhibitors (taxol and vinblastine). CTS induced a greater reduction in ER-mitochondria contacts than these inhibitors (\u003cstrong\u003eSupplementary Fig. 2f\u003c/strong\u003e), indicating that it does not directly disrupt microtubule dynamics at early time points.\u003c/p\u003e\n\u003cp\u003eMAM is crucial for the transport of calcium and lipids between the ER and mitochondria. To assess the impact of CTS on ER-mitochondrial communication, we first evaluated mitochondrial calcium dynamics. Using Rhod-2 fluorescence to monitor mitochondrial calcium, we found that histamine-induced calcium influx (via IP\u003csub\u003e3\u003c/sub\u003eR activation) increased mitochondrial calcium levels in oxLDL-treated cells, which was associated with enhanced ER-mitochondrial interactions. However, this response was significantly reduced in CTS-treated cells, suggesting that CTS disrupts calcium transfer by altering ER-mitochondrial proximity (\u003cstrong\u003eFig. 1e\u003c/strong\u003e). Notably, CTS did not alter p-IP\u003csub\u003e3\u003c/sub\u003eR levels, indicating that ER calcium release was unchanged (\u003cstrong\u003eSupplementary Fig. 2g\u003c/strong\u003e).\u003csup\u003e23\u003c/sup\u003e The reduced mitochondrial calcium levels in CTS-treated cells corresponded to a decrease in ATP production (\u003cstrong\u003eFig. 1f\u003c/strong\u003e). Furthermore, lipid transport between the ER and mitochondria was assessed using nUHPLC-ESI-MS/MS. CTS treatment decreased mitochondrial levels of several phospholipids, including phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylethanolamine (PE), and phosphatidylcholine (PC), suggesting altered lipid transfer between the two organelles (\u003cstrong\u003eFig. 1g\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHolotomography is an advanced imaging technology that captures 3D images of live cells without fluorescence staining and analyzes various cellular parameters based on refractive index measurements.\u003csup\u003e24\u003c/sup\u003e To assess the impact of CTS on intracellular dynamics, we used holotomography to visualize HUVECs after 24 hours of treatment with oxLDL or oxLDL+CTS. This analysis revealed mitochondrial fragmentation and the accumulation of globular, refractile structures indicative of lipid in oxLDL-treated cells, both of which were alleviated by CTS treatment (\u003cstrong\u003eFig. 1h\u003c/strong\u003e). AI-based analysis revealed that oxLDL-treated cells formed distinct clusters, whereas oxLDL+CTS-treated cells exhibited clustering patterns similar to those of control cells depending on the duration of treatment effectively alleviating the morphological changes induced by oxLDL (\u003cstrong\u003eFig. 1i\u003c/strong\u003e). Furthermore, in the presence of oxLDL, CTS treatment reduced ER stress and in CHOP-mCherry reporter cells\u003csup\u003e22\u003c/sup\u003e (\u003cstrong\u003eFig. 1j, Supplementary Fig. 2h\u003c/strong\u003e) and mROS in HUVECs (\u003cstrong\u003eFig. 1k\u003c/strong\u003e), leading to improved cellular health and alleviation of pathological factors associated with atherosclerosis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCTS Enhances Autophagic Flux in Atherosclerosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLysotracker fluorescence intensity increased by approximately 20% in CTS-treated cells, suggesting the restoration of lysosomal function impaired by oxLDL treatment (\u003cstrong\u003eFig. 2a, Supplementary Fig.3a\u003c/strong\u003e). CTS treatment led to an increase in LC3-I to LC3-II conversion levels and promoted both the accumulation and degradation of SQSTM1 over time (\u003cstrong\u003eFig. 2b\u003c/strong\u003e). Co-treatment with CTS and chloroquine (CQ, an autophagy inhibitor), further elevated LC3-II levels compared to CQ alone, demonstrating CTS-induced autophagic flux (\u003cstrong\u003eFig. 2c\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eSince calcium signaling is crucial for autophagy regulation and ER-mitochondria interactions maintain calcium homeostasis, we next focused on cytoplasmic calcium dynamics to explore how reduced ER-mitochondria interactions affect intracellular signaling. In CTS-treated HUVECs, cytosolic calcium levels increased, as shown by Fluo-4 fluorescence (\u003cstrong\u003eFig. 2d\u003c/strong\u003e). This increase was not due to lysosomal calcium release, as confirmed by the use of GCaMP3-TRPML1, a genetically encoded calcium sensor specifically targeting lysosomes.\u003csup\u003e25\u003c/sup\u003e GCaMP3 fluorescence remains unchanged with CTS treatment (\u003cstrong\u003eSupplementary Fig. 3b\u003c/strong\u003e), confirming that CTS alters cytosolic calcium dynamics independently of lysosomal release. Disruption of MAM and the subsequent elevation in cytosolic calcium by CTS induced dose-dependent nuclear translocation of TFEB, as confirmed by immunoblotting and immunofluorescence staining (\u003cstrong\u003eFig. 2e, Supplementary Fig. 3c\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThese findings suggest that\u0026nbsp;CTS treatment promotes autophagic flux through the disruption of the ER-mitochondria interactions, which in turn modulates calcium signaling pathways critical for autophagy regulation, including the activation of TFEB and downstream autophagic markers.\u003c/p\u003e\n\u003cp\u003eTo evaluate the lipid-lowering effects of CTS, DiI-labeled oxLDL-treated HUVECs were analyzed. Co-treatment with bafilomycin A1 (BafA1), an autophagy inhibitor, blocked CTS-mediated lipid reduction, confirming that this process is autophagy-dependent (\u003cstrong\u003eFig. 2f\u003c/strong\u003e). Moreover, CTS significantly reduced lipid levels in wild-type HeLa cells, but had no effect in ATG3 knockout HeLa cells, which are defective in autophagosome formation (\u003cstrong\u003eFig. 2g-h\u003c/strong\u003e). These findings suggest that CTS could serve as a potential therapeutic agent in treating atherosclerosis and other metabolic disorders by enhancing autophagic flux and reducing lipid accumulation in endothelial cells.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCTS Ameliorates Atherosclerosis in ApoE-/- Mice via Autophagy Induction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe next evaluated the efficacy of CTS in modulating autophagy and alleviating atherosclerosis in an ApoE-/- mouse model. CTS (20 mg/kg) was administered intraperitoneally every 2 days for 8 weeks to mice fed a high-cholesterol diet (\u003cstrong\u003eFig. 3a\u003c/strong\u003e). This treatment did not affect body weight (\u003cstrong\u003eFig. 3b\u003c/strong\u003e), indicating low toxicity. Notably, serum total cholesterol and triglyceride levels decreased in CTS-treated mice (\u003cstrong\u003eFig. 3c-d\u003c/strong\u003e), and Oil-Red-O staining revealed a reduction in the aortic root lesion area (\u003cstrong\u003eFig. 3e\u003c/strong\u003e). Furthermore, atherosclerotic plaque formation in the aorta was significantly reduced in CTS-treated mice compared to vehicle-treated controls (\u003cstrong\u003eFig. 3f\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eWe then investigated whether CTS induces autophagy and modulates MAM in vivo. LC3B and SQSTM1 colocalization was assessed in aortic sinus sections, revealing increased intensities of both markers in CTS-treated mice (\u003cstrong\u003eSupplementary Fig. 3d\u003c/strong\u003e). In primary mouse peritoneal macrophages, CTS treatment reversed the decrease in LC3B levels and the increase in lipid content observed in vehicle-treated animals (\u003cstrong\u003eFig. 3g\u003c/strong\u003e). Additionally, PLA assays between IP\u003csub\u003e3\u003c/sub\u003eR and VDAC1 on aortic sinus sections revealed a marked reduction in PLA dot numbers, indicating decreased ER-mitochondria interactions following CTS treatment (\u003cstrong\u003eFig. 3h\u003c/strong\u003e). Similarly, PLA performed on peritoneal macrophages of CTS-treated mice showed a decrease in PLA dot numbers (\u003cstrong\u003eFig. 3i\u003c/strong\u003e). These findings suggest that CTS, through the modulation of autophagy and disruption of ER-mitochondrial interactions, holds promise as a targeted therapeutic strategy to alleviate atherosclerosis by reducing lipid accumulation and plaque formation in vascular tissues.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIdentification of HYOU1 as a Target of CTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDrug affinity responsive target stability (DARTS)-LC-MS/MS is a powerful technique for identifying small molecule targets without requiring chemical modification,\u003csup\u003e26, 27\u003c/sup\u003e and has been previously employed to identify targets of autophagy-regulating compounds.\u003csup\u003e28-30\u003c/sup\u003e Using this approach, we aimed to identify CTS target proteins involved in ER-mitochondria interactions and autophagy, with a particular focus on ER-localized proteins due to their central role in stress response and autophagy regulation. Membrane proteomes were treated with CTS and analyzed by DARTS-LC-MS/MS (\u003cstrong\u003eFig. 4a\u003c/strong\u003e). SWATH analysis quantified 872 proteins, with 329 exhibiting a coefficient of variation (CV) of less than 20%. Among these, 69 proteins were degraded by pronase (\u0026gt;10%), while 16 proteins were stabilized by CTS binding (FC \u0026ge; 1.2, \u003cstrong\u003eFig. 4b\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eCandidate proteins were predominantly localized to the ER or mitochondria (\u003cstrong\u003eFig. 4c\u003c/strong\u003e), with STRING analysis linking them to key processes including ER stress (HYOU1 and CANX), vesicle trafficking (RAB1A and RAB10), and metabolism (ALDH18A1, HSD17B10, and DLAT) (\u003cstrong\u003eFig. 4d\u003c/strong\u003e). Our focus was on ER-localized candidates (\u003cstrong\u003eFig. 4e\u003c/strong\u003e), given the ER\u0026apos;s central role in stress response and autophagy regulation. DARTS-Western blot analysis of HUVECs lysates confirmed that HYOU1 showed resistance to degradation by pronase in the presence of CTS binding, demonstrating a clear binding pattern. In contrast, RAB10 and CANX did not show a similar pattern, further supporting the specificity of CTS-HYOU1 interaction (\u003cstrong\u003eSupplementary Fig. 4a-b\u003c/strong\u003e). LC-MS/MS analysis identified 16 peptide fragments corresponding to HYOU1, along with 7 peptides in the NBD and SBD domains, which exhibited reduced degradation upon pronase treatment (\u003cstrong\u003eSupplementary Fig. 4c\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDose-dependent DARTS analysis revealed an EC50 of approximately 1 \u0026micro;M for CTS binding to HYOU1 (\u003cstrong\u003eFig. 4f\u003c/strong\u003e). Further validation via a cellular thermal shift assay (CETSA) demonstrated that CTS prevented thermal denaturation of HYOU1 in live HEK293 cells (\u003cstrong\u003eFig. 4g\u003c/strong\u003e). Full-length HYOU1 constructs were cloned into the pEZT-BM vector\u003csup\u003e31\u003c/sup\u003e and the HYOU1(1-912) protein was expressed and purified (\u003cstrong\u003eSupplementary Fig. 4d\u003c/strong\u003e). Microscale thermophoresis (MST) analysis of CTS binding to HYOU1 showed a dissociation constant (Kd) of 1.42 \u0026micro;M, confirming direct interaction between CTS and HYOU1 (\u003cstrong\u003eFig. 4h\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIdentification of Critical Binding Sites for CTS on HYOU1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate potential binding sites for CTS on HYOU1, we employed in silico docking analysis using the Alphafold 3D structure of HYOU1 (\u003cstrong\u003eFig. 5a\u003c/strong\u003e). CTS was predicted to bind a pocket within the NBD of HYOU1, with residues R217 and N410 likely interacting with CTS via hydrogen bonds (\u003cstrong\u003eFig. 5b\u003c/strong\u003e). We experimentally verified these binding sites by generating point mutations: HYOU1-R217A and N410D. DARTS analysis of HEK293 cells transfected with MYC-tagged wild-type (WT) or mutant HYOU1 plasmids revealed that CTS could not protect the mutant HYOU1 from proteolysis, confirming the critical role of these residues in CTS binding (\u003cstrong\u003eFig. 5c\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eMoreover, in HUVECs expressing mutant HYOU1, CTS treatment led to reduced autophagosome formation, as assessed by LC3B immunofluorescence staining (\u003cstrong\u003eFig. 5d\u003c/strong\u003e), highlighting the importance of R217 and N410 in CTS-mediated autophagy regulation. Collectively, we identified HYOU1 as a target of CTS through DARTS-LC-MS/MS and validated this interaction using \u003cem\u003ein silico\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e assays.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHYOU1 as a Key Mediator of CTS-Regulated ER-Mitochondrial Interactions and Autophagy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGlucose-regulated proteins (GRPs), localized in the ER and mitochondria, function as chaperones that maintain organelle homeostasis.\u003csup\u003e18\u003c/sup\u003e Specifically, GRP75 forms the IP\u003csub\u003e3\u003c/sub\u003eR-GRP75-VDAC1 complex, contributing to ER-mitochondrial coupling, while GRP78 interacts with IP\u003csub\u003e3\u003c/sub\u003eR1 to facilitate ER calcium release.\u003csup\u003e32\u003c/sup\u003e Although HYOU1 (also known as GRP170) shares structural and functional similarities with GRP75 and GRP78,\u003csup\u003e18\u003c/sup\u003e it has not been as extensively studied. Therefore, we hypothesized that HYOU1 may play a distinct role in regulating MAM and autophagy.\u003c/p\u003e\n\u003cp\u003eWe examined how CTS-mediated regulation of HYOU1 affects ER-mitochondria interactions. We speculated that HYOU1 might indirectly modulate the IP\u003csub\u003e3\u003c/sub\u003eR-GRP75-VDAC1 complex, potentially involving GRP78 in this interaction network (\u003cstrong\u003eFig. 6c\u003c/strong\u003e). PLA results showed that CTS treatment for 4 hours reduced interactions between HYOU1-IP\u003csub\u003e3\u003c/sub\u003eR, HYOU1-GRP78, and IP\u003csub\u003e3\u003c/sub\u003eR-GRP78, which led to the dissociation of IP\u003csub\u003e3\u003c/sub\u003eR-GRP75, GRP75-VDAC1, and IP\u003csub\u003e3\u003c/sub\u003eR-VDAC1 complexes (\u003cstrong\u003eFig. 6a\u003c/strong\u003e). Co-immunoprecipitation further confirmed the reduced interaction between HYOU1 and IP\u003csub\u003e3\u003c/sub\u003eR in CTS-treated cells (\u003cstrong\u003eFig. 6b\u003c/strong\u003e), suggesting that CTS binding induces a conformational change in HYOU1, decreasing its affinity for IP\u003csub\u003e3\u003c/sub\u003eR (\u003cstrong\u003eFig. 6c\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eA recently developed HYOU1 inhibitor (HYOU1-IN),\u003csup\u003e20\u003c/sup\u003e intended for fibroblast activation suppression, was found to modulate MAM dynamics (\u003cstrong\u003eSupplementary Fig. 5a-b\u003c/strong\u003e). In addition, HYOU1-IN disrupted the interaction between HYOU1 and GRP78 (\u003cstrong\u003eSupplementary Fig. 5c\u003c/strong\u003e), suggesting that this interaction is critical for MAM complex stability. Notably, CTS exhibited greater potency than HYOU1-IN in modulating MAM dynamics. These findings demonstrate a functional link between HYOU1 and MAM homeostasis, providing mechanistic insights into its role in disease progression and underscoring its potential as a therapeutic target.\u003c/p\u003e\n\u003cp\u003eWe next examined whether HYOU1 mediates the biological effects of CTS by assessing MAM formation following HYOU1 knockdown. In HYOU1-depleted HUVECs, ER-mitochondria contacts were disrupted (\u003cstrong\u003eFig. 6d\u003c/strong\u003e), cytosolic calcium levels increased (\u003cstrong\u003eFig. 6e\u003c/strong\u003e), and autophagic flux was enhanced, as indicated by increased LC3-II conversion and reduced SQSTM1 (\u003cstrong\u003eFig. 6f\u003c/strong\u003e). Additionally, lipid levels were reduced in these cells (\u003cstrong\u003eFig. 6g\u003c/strong\u003e), and oxLDL-induced foam cell formation was inhibited in HYOU1-deficient macrophages (\u003cstrong\u003eFig. 6h\u003c/strong\u003e), suggesting a role for HYOU1 in atherosclerosis progression.\u003c/p\u003e\n\u003cp\u003eTo confirm that HYOU1 is a critical target of CTS, we generated HYOU1 heterozygous knockout (KO) cells using CRISPR-Cas9 (\u003cstrong\u003eFig. 7a, Supplementary Fig. 6a-b\u003c/strong\u003e).\u003csup\u003e33, 34\u003c/sup\u003e PLA assays revealed diminished ER-mitochondria contact in HYOU1 KO cells compared to WT cells (\u003cstrong\u003eFig. 7b\u003c/strong\u003e), indicating that HYOU1 is essential for maintaining MAM integrity. Upon CTS treatment, the effects on ER-mitochondria contacts (\u003cstrong\u003eFig. 7c-d\u003c/strong\u003e), lipid levels (\u003cstrong\u003eFig. 7e\u003c/strong\u003e), and other CTS-induced changes were reduced in HYOU1 KO cells compared to WT cells (\u003cstrong\u003eSupplementary Fig. 6c-d\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAssociation of HYOU1 with Atherosclerosis Progression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess the relevance of our mechanistic findings in human disease, we analyzed single-cell RNA sequencing (scRNA-seq) data from human carotid atherosclerotic plaques (AC) and adjacent tissue (PA) from patients undergoing carotid endarterectomy (GSE159677; n=3 per group).\u003csup\u003e35\u003c/sup\u003e Unsupervised clustering identified 10 major cell populations, including monocytes, dendritic cells, T cells, endothelial cells, smooth muscle cells, fibroblasts, B cells, plasma cells, mast cells, and NK cells (\u003cstrong\u003eFig. 7f\u003c/strong\u003e). We visualized gene expression across these clusters using FeaturePlot analysis. Genes involved in MAM formation (HYOU1, GRP75/HSPA9, GRP78/HSPA5) (\u003cstrong\u003eFig. 7g-i\u003c/strong\u003e), autophagy regulation (RUBCN, MTOR) (\u003cstrong\u003eSupplementary Fig. 7a-b\u003c/strong\u003e), and lipid metabolism (CD36) (\u003cstrong\u003eFig. 7j\u003c/strong\u003e) were more prominently expressed in AC compared to PA tissues, especially in monocytes and dendritic cells. FKBP2, a gene highly correlated with HYOU1 in the Human Protein Atlas scRNA-seq dataset (r = 1.000), was similarly enriched in the AC condition (\u003cstrong\u003eSupplementary Fig.\u003c/strong\u003e \u003cstrong\u003e7c\u003c/strong\u003e), suggesting its involvement in the HYOU1-associated regulatory network.\u003c/p\u003e\n\u003cp\u003eTo quantify expression differences between disease and control, we performed pseudobulk analysis. This approach confirmed increased expression of HYOU1, GRP75, GRP78, FKBP2, CD36, RUBCN, and MTOR in AC tissues, particularly in monocytes and dendritic cells (\u003cstrong\u003eFig. 7k-l\u003c/strong\u003e). These data indicate a disease-associated upregulation of genes linked to MAM integrity, autophagy, and lipid handling. Together, these single-cell transcriptomic analyses reinforce the pathological relevance of the HYOU1-dependent gene network in atherosclerotic immune cell populations and support its role in autophagy suppression and metabolic dysfunction in vascular inflammation.\u003c/p\u003e\n\u003cp\u003eWe investigated the potential association between HYOU1 and disease progression by analyzing a proteomics dataset of monocytes from atherosclerosis patients (PXD041367) in the PRIDE database (https://www.ebi.ac.uk/pride/).\u003csup\u003e36\u003c/sup\u003e Our analysis revealed that HYOU1 expression was upregulated in patients compared to normal controls and increased as the disease progressed (\u003cstrong\u003eFig. 7m\u003c/strong\u003e). Notably, HYOU1 levels positively correlated with CD36 (\u003cstrong\u003eFig. 7n\u003c/strong\u003e), which was also upregulated in the disease group in single-cell RNA sequencing analysis (\u003cstrong\u003eFig. 7j\u003c/strong\u003e).\u0026nbsp;Furthermore, proteomics analysis revealed a positive correlation between HYOU1 and GRP78 protein levels (\u003cstrong\u003eFig. 7o\u003c/strong\u003e), consistent with findings from single-cell RNA sequencing analysis (\u003cstrong\u003eFig. 7i\u003c/strong\u003e). These results suggest that these ER chaperones may be co-regulated in the context of atherosclerosis, highlighting the potential role of ER stress in disease progression through the coordinated regulation of HYOU1 and GRP78.\u003c/p\u003e\n\u003cp\u003eIn summary, our results identify HYOU1 as a novel regulator of the IP\u003csub\u003e3\u003c/sub\u003eR-GRP75-VDAC1 complex in the MAM, which plays a critical role in controlling autophagy and mitigating autophagy-related disease phenotypes, particularly atherosclerosis (\u003cstrong\u003eFig. 8\u003c/strong\u003e). Targeting HYOU1 may therefore represent a promising therapeutic strategy for treating related diseases.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eHere, we uncover a novel link between HYOU1, MAM regulation, autophagy, and atherosclerosis. Although organelle interactions are increasingly recognized as key regulators of cellular homeostasis, few studies have explored their therapeutic modulation in human disease. Our findings demonstrate that cryptotanshinone (CTS), a natural anti-atherosclerotic compound, directly targets HYOU1 to rewire ER-mitochondria communication and enhance autophagy. This work positions HYOU1 as a critical regulator of MAM stability and highlights CTS as a promising therapeutic agent for metabolic disorders (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ep). Using a label-free target identification approach, we identified HYOU1 as the biological target of CTS. CTS binds the nucleotide-binding domain (NBD) of HYOU1, disrupting its interaction with GRP78 and IP\u003csub\u003e3\u003c/sub\u003eR, leading to the dissociation of the IP\u003csub\u003e3\u003c/sub\u003eR-GRP75-VDAC1 complex at the MAM. This reduces excessive calcium transfer from the ER to mitochondria, alleviating mitochondrial overload and ATP depletion.\u003c/p\u003e \u003cp\u003eMitochondrial calcium overload is a known driver of mROS production, which further exacerbates ER stress, lipid peroxidation, and inflammatory signaling.\u003csup\u003e\u003cspan additionalcitationids=\"CR38 CR39 CR40\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e Autophagy dysfunction amplifies this oxidative stress, as impaired clearance of damaged mitochondria leads to sustained mROS accumulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ep, upper panel). This creates a vicious cycle where mROS further suppresses autophagy and promotes atherosclerosis progression. Our findings suggest that CTS indirectly reduces mROS levels by restoring autophagy, rather than acting as a direct antioxidant. By preserving mitochondrial quality control mechanisms, CTS prevents excessive mROS production and mitigates oxidative damage.\u003c/p\u003e \u003cp\u003eMolecular modeling suggests that CTS interacts with R217 and N410 in HYOU1\u0026rsquo;s NBD. Mutagenesis studies confirmed that these residues are critical for CTS binding and autophagy activation, as CTS failed to protect mutant HYOU1 from proteolysis and did not effectively induce autophagosome formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec-d). Recent studies indicate that ATP binding to HYOU1 induces a conformational change that enhances its interaction with GRP78, with the N410 residue near the ATP binding site playing a critical role in this interaction.\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e CTS binds to N410, allosterically modulating ATP binding and destabilizing the HYOU1-GRP78 complex, potentially influencing its interaction with IP\u003csub\u003e3\u003c/sub\u003eR. These findings establish HYOU1 as a key regulator of ER-mitochondria crosstalk, autophagy, and oxidative stress balance, further supporting its role in the atherosclerotic cycle (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ep).\u003c/p\u003e \u003cp\u003eAs illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ep (lower panel), our study reveals that HYOU1-dependent MAM regulation plays a key role in the atherosclerosis. The continuous loop of oxLDL exposure, ER stress, excessive mitochondrial calcium influx, ROS accumulation, and autophagy impairment drives atherosclerosis progression. Our findings suggest that CTS breaks this pathological cycle by restoring MAM homeostasis, reducing calcium overload, and enhancing autophagy, ultimately mitigating atherosclerosis development.\u003c/p\u003e \u003cp\u003eAlthough our experimental models were limited to in vitro and murine systems, we analyzed publicly available single-cell RNA sequencing data from human atherosclerotic plaques to assess the clinical relevance of our findings. The observed upregulation of HYOU1 and its associated regulators\u0026mdash;GRP75, GRP78, FKBP2, RUBCN, MTOR, and CD36\u0026mdash;was most prominent in monocytes, where both differential expression between diseased and normal tissues and co-expression with HYOU1 were clearly observed. This cell-type-specific enrichment aligns with the established role of monocytes in lipid uptake, inflammatory signaling, and foam cell formation during atherosclerosis.\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e While these observations are correlative and do not establish causality, they are consistent with our mechanistic model and support the translational relevance of the HYOU1-centered MAM-autophagy network in human disease.\u003c/p\u003e \u003cp\u003eBeyond atherosclerosis, MAM dysfunction contributes to ferroptosis, lipid metabolism disorders, and cancer. Zhang, Z et al. recently identified a ferroptosis inhibitor that targets the sigma-1 receptor (Sig1R), an ER chaperone, demonstrating that MAM regulation can influence ferroptotic cell death.\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e Inspired by these findings, we investigated whether CTS could similarly modulate ferroptosis via MAM regulation. In RSL3 (ferroptosis inducer)-treated HUVECs, CTS simultaneously promoted cell survival and reduced ER-mitochondria tethering, suggesting a potential protective effect (\u003cb\u003eSupplementary Fig.\u0026nbsp;8a-b\u003c/b\u003e). Lipidomics analysis further revealed that CTS decreases polyunsaturated phospholipid content, particularly mitochondrial PE 38:4, PE 40:4, and PS 36:4 suggesting a mechanism for its anti-ferroptotic effects (\u003cb\u003eSupplementary Fig.\u0026nbsp;8c-e\u003c/b\u003e). These findings not only strengthen the link between ferroptosis and MAM, but suggest that the discovery of MAM-modulating compounds represents a promising strategy for regulating intracellular homeostasis and stress responses. Given the critical role of inflammation in atherosclerosis, we also investigated CTS's effect on T cell activation. We observed that CTS inhibited the MAM formation induced by TCR stimulation in human CD4 T cells. Notably, this was accompanied by a reduction in the expression of T cell activation markers (such as CD44, CD25). These findings suggest a potential link between MAM regulation and T cell activation in the context of the inflammatory response. While the exact mechanism remains to be further explored, these results imply that CTS may modulate MAM dynamics to influence T cell activation, offering new insights into the regulation of inflammation in atherosclerosis.\u003c/p\u003e \u003cp\u003eNumerous studies have shown that HYOU1 is highly expressed in various diseases, particularly in cancer. In epithelial ovarian cancer and lung cancer, HYOU1 drives tumor progression and represents a key molecular target with prognostic significance.\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e Consistent with these findings, we observed that HYOU1 knockout (KO) cells exhibit a slower proliferation rate compared to wild-type (WT) cells (\u003cb\u003eSupplementary Fig.\u0026nbsp;6e\u003c/b\u003e), further supporting its role in cell survival and growth regulation. These observations suggest that HYOU1 may function as a key metabolic regulator across multiple disease contexts. While further studies are needed to determine the molecular pathways linking HYOU1 to tumorigenesis, its expression profile highlights its potential as a biomarker and therapeutic target for cancer and metabolic disorders.\u003c/p\u003e \u003cp\u003eAs summarized in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ep, HYOU1 serves as a central regulator of ER-mitochondrial communication and autophagy, modulating cellular stress responses under conditions resembling oxLDL-induced atherosclerosis. CTS directly interacts with the NBD of HYOU1, disrupting MAM-associated calcium transfer, thereby restoring autophagic homeostasis and alleviating lipid accumulation. These findings position HYOU1 as a promising therapeutic target, with CTS-based interventions offering a novel strategy to break the oxLDL-driven atherosclerotic cycle and regulate organelle crosstalk in disease.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eCell Culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman umbilical vein endothelial cells (HUVECs, up to passage 9) were purchased from Lifeline Cell Technology (Frederick, MD) and cultured in EBM-2 Basal Medium supplemented with growth factors. HEK293, HepG2, HeLa, and RAW264.7 cells were purchased from the Korean Cell Bank (Seoul, South Korea) and cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics. All cell cultures were maintained at 37\u0026deg;C in a humidified incubator with 5% CO2 and pH 7.4. Cells were tested for mycoplasma contamination every two months.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCRISPR-Cas9 Knockout Cell Generation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTarget sequences for single Single-guide RNAs (sgRNAs) were designed using the Cas-Designer (http://www.rgenome.net/cas-designer/). Each sgRNA spacer oligo was cloned into the pRG2 vector, and plasmid sequences were verified by Sanger sequencing. HEK293T cells were transfected with 375 ng of Cas9 and 125 ng of sgRNA-expressing plasmids using the jetOPTIMUS transfection reagent, following the manufacturer\u0026rsquo;s protocol. Single cells were isolated by limiting dilution, and knockout clones were validated using next-generation sequencing (NGS) with the Illumina MiniSeq system. The NGS data were analyzed using Cas-Analyzer (http://www.rgenome.net/cas-analyzer/).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were conducted in accordance with the ethical guidelines approved by the Institutional Animal Care and Use Committee of Yonsei University (IACUC-A-202208-1519-01).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimal Studies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eApoE\u003csup\u003e-/-\u0026nbsp;\u003c/sup\u003eC57BL/6J male mice were maintained in a specific-pathogen-free facility under a 12-hour light/dark cycle with ad libitum access to food and water. Mice (n = 6) were randomly assigned to experimental groups, with body weight matched across groups at the time of allocation. Mice were fed a high-cholesterol diet (Research Diets Inc., New Brunswick, NJ) starting at 6 weeks of age and continued until the conclusion of the study, with the final sacrifice performed at 18 weeks of age. For intraperitoneal injections, the vehicle solution consisted of 30% polyethylene glycol 300 and 5% Tween 80 in saline. At the experimental endpoint, mice were anesthetized using avertin.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePLA (Proximity Ligation Assay)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were grown on a chambered cell culture slide (SPL Life Sciences, Pocheon, South Korea) and treated with the drug. PLA experiments were performed using the Duolink In Situ Red Kit or NaveniFlex Cell MR Red kit according to manufacturer instructions. Briefly, cells were fixed with 4% paraformaldehyde for 10 minutes and permeabilized with 0.2% Triton X-100 for 20 minutes at room temperature. The cells were then blocked with blocking solution for 1 hour and incubated with primary antibodies overnight at 4\u0026deg;C. PLA probes were applied and incubated for 1 hour at 37\u0026deg;C. Cells were incubated with ligase for 30 minutes, followed by DNA polymerase incubation for 100 minutes. Samples were mounted with mounting medium containing DAPI. PLA signals were observed using an LSM 980 confocal microscope. Quantification of PLA signals was performed by counting dots or measuring fluorescence intensity per cell area.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCalcium Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo monitor mitochondrial or cytosolic calcium influx, HUVECs were seeded onto chambered coverslips (ibidi, Gr\u0026auml;felfing, Germany, #80827) and incubated with either Rhod-2 AM or Fluo-4 AM in Krebs-Ringer-HEPES (KRH) buffer (pH 7.4) for 30 minutes at 37\u0026deg;C incubator. After washing with KRH buffer, the cells were incubated in calcium-free KRH buffer (pH 7.4) for an additional 30 minutes to allow for the de-esterification of the dyes. Calcium influx was assessed by capturing live-cell images using an LSM980 confocal microscope. Fluorescence intensity within selected regions of interest (ROIs) was quantified over time using ImageJ software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTransmission Electron Microscopy (TEM)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHUVECs, HEK293T control cells, and HEK293T HYOU1 knockout (KO) cells were cultured in culture dishes (NEST Biotechnology, Wuxi, China) until they reached 50\u0026ndash;60% confluence. Cells were treated with 5 \u0026micro;M CTS or dimethyl sulfoxide (DMSO) as a vehicle control for 24 hours at 36\u0026deg;C. After treatment, cells were fixed in a solution containing 1% glutaraldehyde and 1% paraformaldehyde in 0.3 M cacodylate buffer for 1 hour at 4\u0026deg;C. Fixed samples were post-fixed in 1% osmium tetroxide in distilled water for 1 hour at 4\u0026deg;C. The specimens were then stained with 1% uranyl acetate overnight at 4\u0026deg;C, dehydrated through a graded ethanol series, and embedded in epoxy resin (EMS, Hatfield, PA). Ultrathin sections (~60 nm) were prepared using an ultramicrotome (Leica Microsystems, Wetzlar, Germany) and mounted on copper grids. Sections were stained with UranyLess for 2 minutes, rinsed with distilled water, and counterstained with lead citrate for 1 minute. Transmission electron microscopy (TEM) images were acquired using a Tecnai 20 transmission electron microscope (Thermo Fisher Scientific, Waltham, MA) operating at 120 kV, equipped with a US1000XP camera.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHolotomography\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHUVECs were seeded onto HT-Ready 96 well plate (Tomocube, Daejeon, Republic of Korea). The cells were then treated with either oxLDL alone or a combination of oxLDL and CTS for 24 hours. High-resolution live cell images were acquired using HT-X1 holotomographic microscope (Tomocube), which utilizes low-coherence light sources. During imaging, the plate was maintained in a wet chamber with controlled temperature (37\u0026deg;C) and CO2 levels (5%). Images were analyzed using AI-based TomoAnalytic 2.0 software (Tomocube).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLipidomics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHUVECs treated with or without 5 \u0026micro;M CTS were harvested, and mitochondria were isolated using a mitochondria isolation kit according to the manufacturer\u0026rsquo;s instructions. A portion of the mitochondrial pellet was resuspended in 2% CHAPS in Tris-buffered saline (pH 7.2), and protein concentration was determined using a BCA assay. The remaining pellet was stored at -80\u0026deg;C for subsequent lipidomic analysis. Detailed methods are provided in the \u003cem\u003esupplementary material.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDARTS-LC-MS/MS Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMembrane proteins were extracted from HepG2 and HUVEC cells using a Membrane Protein Extraction Kit, and protein concentration was adjusted to 1 mg/mL. All steps were performed on ice to minimize protein degradation. Protein samples were incubated with either DMSO or CTS at 4\u0026deg;C for 3 hours with gentle rocking. After incubation, pronase was added to each sample, and digestion was carried out at room temperature for 10 minutes. The reaction was stopped by adding 6\u0026times; SDS sample buffer, followed by heat denaturation. The digested protein samples were separated by SDS-PAGE, and in-gel digestion was performed. Peptides were analyzed using a Triple TOF 6600 mass spectrometer (AB Sciex, Concord, Ontario, Canada) coupled with an Acquity UPLC system (Waters, Milford, MA). Both information-dependent acquisition (IDA) and sequential window acquisition of all theoretical fragment-ion spectra (SWATH) tandem mass spectrometry methods were applied.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCETSA analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHEK293 cells were harvested using trypsin-EDTA solution. Cell suspensions at a concentration of 3\u0026times;107 cells/15 mL was aliquoted into each conical tube, CTS was added, with DMSO used as a control. The cells were incubated for 1 hour in a CO2 incubator at 37 \u0026deg;C with gently mixing. After centrifugation, the pellet was washed with PBS and resuspended in 1 mL of PBS (containing protease inhibitors). The suspension was aliquoted into PCR tubes (~100 \u0026mu;L per tube) and heated between 40-64\u0026deg;C for 3 minutes, followed by cooling to 25\u0026deg;C for an additional 3 minutes in a thermal cycler. The tubes were then centrifuged to pellet the cells and the supernatant was discarded. The pellet was resuspended in 0.4% NP-40 (in PBS) supplemented with protease inhibitors to facilitate the dissolution of hydrophobic proteins. The cell suspension was subjected to two cycles of freezing-thawing in liquid nitrogen and centrifuged at 20,000 g for 20 min at 4 \u0026deg;C. The supernatants (soluble proteins) were collected and used for western blotting.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMicroscale thermophoresis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFluorescence labeling of purified HYOU1 protein was performed using the Protein Labeling Kit RED-NHS (NanoTemper Technologies, Munich, Germany), according to the manufacturer\u0026rsquo;s protocol. Binding assays were performed using the Monolith NT.115pico instrument (NanoTemper Technologies). Labeled protein was prepared at a final concentration of 20 nM in assay buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl) supplemented with 0.05% Tween-80 to minimize protein aggregation. The labeled protein was mixed with serially diluted ligand samples and loaded into premium capillaries (NanoTemper Technologies). Thermophoresis was measured and the dissociation constant (Kd) was determined. Data analysis was performed using MO Affinity Analysis software (NanoTemper Technologies).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn silico\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;docking study\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMolecular docking analysis was performed using Discovery Studio 2018 software (BIOVIA, San Diego, CA). The 3D structure of HYOU1 was obtained from the Alphafold database, and the ligand structure was obtained from the Pubchem. For the docking simulations, the CDOCKER method, a grid-based docking technique, was employed to predict the binding mode of the ligands to the target protein. The Binding sites on protein were defined based on receptor\u0026rsquo;s cavities, then the ligand was docked into these binding sites. The top 10 docking poses were generated based on the binding affinity, and the binding energy (CDOCKER energy) for each pose was calculated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003escRNA-seq data analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSingle-cell RNA sequencing (scRNA-seq) data were analyzed using Seurat (version 5) in R Studio(V 4.4.1).\u003csup\u003e47, 48\u003c/sup\u003e Seurat objects were created for each sample, with PA tissues labeled as Normal and AC plaques as Disease. After merging, QC filtering retained cells with \u0026gt;200 but \u0026lt;6,000 detected genes and \u0026lt;5% mitochondrial reads. The data were normalized using the SCTransform method,\u003csup\u003e49\u003c/sup\u003e and highly variable genes were identified. Principal component analysis (PCA) was used for dimensionality reduction, and the top components were used for shared nearest neighbor (SNN)-based clustering (resolution = 0.8) and UMAP visualization.. Harmony was applied for batch correction. Cell type annotation was based on marker genes identified using FindAllMarkers (min.pct = 0.25, logFC \u0026gt; 0.25) and assigned to known cell types, including T cells, monocytes, dendritic cells, endothelial cells, smooth muscle cells, fibroblasts, plasma cells, mast cells, and natural killer cells.\u003csup\u003e35, 50\u003c/sup\u003e Pseudobulk analysis was performed to aggregate scRNA-seq data at the sample level for bulk-like differential expression analysis using Seurat and DESeq2.\u003csup\u003e47\u003c/sup\u003e Gene expression counts were summed by sample ID and cell type, and raw counts were normalized using variance-stabilizing transformation (VST) in DESeq2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSix mice per group were used in this study. Sample sizes were determined based on institutional guidelines, with a minimum of five mice per group to ensure consistent and reliable results across the experimental groups. No statistical method was used to predetermine the sample size. Since the researchers had to identify the cages of the mice for treatments, blinding could not be applied.\u003c/p\u003e\n\u003cp\u003eAll the data were analyzed using Graph Pad Prism 9.0 (GraphPad Software Inc., San Diego, CA) and expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Comparison between two groups was done by the unpaired Students\u0026rsquo; t-test. Minimum of three independent experiments were conducted to allow for statistical comparisons. P-values less than 0.05 were considered statistically significant (* p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001, ****p\u0026lt;0.0001).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSingle-cell transcriptome profiles of human calcified atherosclerotic core (AC) plaques and patient-matched proximal adjacent (PA) portions of carotid artery tissue were obtained from the Gene Expression Omnibus (GEO) database (GSE159677, PMID: 36224302, DOI: 10.1038/s42003-022-04056-7). \u003csup\u003e35, 50\u003c/sup\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank Dongjin Lee (Yonsei University, Republic of Korea) for preparing DARTS samples for MS analysis. We also express our gratitude to Dr. Dong-Hyung Cho (Kyungpook National University, Republic of Korea) for providing the SPLICS vector, Dr. Hae-Ryung Park (University of Rochester, USA) for supplying the CHOP-mCherry reporter cells, and Dr. Seong-Woon Yu (DIGIST, Republic of Korea) for supplying the ATG3 knockout HeLa cells. We are also grateful to Dr. Goo Taeg Oh (Ewha Womans University, Republic of Korea) for generously gifting us ApoE-/- mice. We also thank Dr. Sumin Lee and Dr. Hyejin Kim (Tomocube, Inc., Republic of Korea) for their assistance with holotomography imaging and AI-based analysis. We also thank Dr. Eun-woo Lee (KRIBB, Republic of Korea) for providing the reagent (RSL3) and offering advice for related experiments. This work was partly supported by grants from the National Research Foundation of Korea, funded by the government of the Republic of Korea: 2021R1A3B1077371 (H.J.K.), 2022R1A2C1009376 (J.Y.M.), 2021R1C1C1012076 (J.Y.), 2021M3E5E3080529 (J.Y.), 2022R1A2C2093050 (M.L.), 2021M3A9H3015389 (S.B.), 2022M3H9A2096186 (J.Y.K.), 2021R1A2C2003171 (M.H.M.) and the Brain Korea 21 Plus Project (H.J.K.). This work was also supported by the ICONS (Institute of Convergence Science, Yonsei University) (H.J.K.), the Research Program of the National Research Council of Science \u0026amp; Technology CRC22021-100 (J.Y.K), and the Korea Basic Science Institute Research Grant No. A423200 (J.Y.K.).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM.K. and H.J.K. planned and conceptualized the study. M.K. performed most experiments with contributions from J.H., S.K., H.E.L., J.Y.M., D.Y., J.Y., H.C., M.L., Y.L., S.B., S.S., J.Y.L., J.Y.K., and M.H.M. M.K. and H.J.K. analyzed data, performed statistics, and wrote the manuscript. All authors have read and approved the article.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWeber, C. \u0026amp; Noels, H. 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Oxidized low-density lipoprotein (oxLDL), a major contributor to atherosclerosis, disrupts cellular homeostasis by promoting the formation of mitochondria-associated ER membrane (MAM), which regulate essential processes such as calcium transport, lipid metabolism, mitochondrial dynamics, and autophagy\u0026mdash;critical components in atherosclerotic signaling pathways. Here, we identify hypoxia-upregulated protein 1 (HYOU1) as a novel regulator of MAM stability, coordinating ER-mitochondrial calcium flux and lipid metabolism. Using DARTS-LC-MS/MS, we demonstrate that cryptotanshinone (CTS), a natural anti-atherosclerotic compound derived from Danshen (\u003cem\u003eSalvia miltiorrhiza\u003c/em\u003e), binds directly to the nucleotide-binding domain (NBD) of HYOU1, disrupting ER-mitochondria interactions. This results in three key outcomes: (1) increased cytosolic calcium levels and TFEB nuclear translocation, (2) decreased mitochondrial calcium influx and ATP production, and (3) enhanced autophagy, which reduces intracellular lipid accumulation. Consequently, CTS reduces mitochondrial ROS accumulation and lipid overload \u003cem\u003ein vitro\u003c/em\u003e, while alleviating atherosclerotic burden in ApoE-/- mice fed a high-cholesterol diet. Single-cell transcriptome analysis of human carotid atherosclerotic plaques further revealed that HYOU1 and its associated autophagy and lipid metabolism regulators are upregulated in monocytes and dendritic cells, underscoring their clinical relevance and co-regulated expression in atherosclerotic lesions. These findings provide the first evidence that HYOU1 as a promising therapeutic target for modulating MAM-autophagy crosstalk and highlight CTS as a potential small-molecule intervention to disrupt the oxLDL-driven atherosclerotic cycle.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e","manuscriptTitle":"Cryptotanshinone Targets HYOU1 to Rewire ER-Mitochondria Communication and Enhance Autophagy in Atherosclerosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-28 02:56:52","doi":"10.21203/rs.3.rs-6301868/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3711be98-7853-45f4-afe1-b749523cc9a0","owner":[],"postedDate":"March 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":46171601,"name":"Cell Communication and Signaling"}],"tags":[],"updatedAt":"2025-04-02T01:35:22+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-28 02:56:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6301868","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6301868","identity":"rs-6301868","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}