ER membrane remodeling by targeting RTN4 induces pyroptosis to facilitate antitumor immune.

Pyroptosis is an identified programmed cell death that has been highly linked to endoplasmic reticulum (ER) dynamics. However, the crucial proteins for modulating dynamic ER membrane curvature change that trigger pyroptosis are currently not well understood. In this study, a biotin-labeled chemical probe of potent pyroptosis inducer α-mangostin (α-MG) was synthesized. Through protein microarray analysis, reticulon-4 (RTN4/Nogo), a crucial regulator of ER membrane curvature, was identified as a target of α-MG. We observed that chemically induced proteasome degradation of RTN4 by α-MG through recruiting E3 ligase UBR5 significantly enhances the pyroptosis phenotype in cancer cells. Interestingly, the downregulation of RTN4 expression significantly facilitated a dynamic remodeling of ER membrane curvature through a transition from tubules to sheets, consequently leading to rapid fusion of the ER with the cell plasma membrane. In particular, the ER-to-plasma membrane fusion process is supported by the observed translocation of several crucial ER markers to the "bubble" structures of pyroptotic cells. Furthermore, α-MG-induced RTN4 knockdown leads to PKM2-dependent conventional caspase-3/GSDME cleavages for pyroptosis progression. In vivo, we observed that chemical or genetic RTN4 knockdown significantly inhibited cancer cells growth, which further exhibited an antitumor immune response with anti-PD-1. In translational research, RTN4 high expression was closely correlated with the tumor metastasis and death of patients. Taken together, RTN4 plays a fundamental role in inducing pyroptosis through the modulation of ER membrane curvature remodeling, thus representing a prospective druggable target for anticancer immunotherapy.

Role of a holo-insertase complex in the biogenesis of biophysically diverse ER membrane proteins.

Mammalian membrane proteins perform essential physiologic functions that rely on their accurate insertion and folding at the endoplasmic reticulum (ER). Using forward and arrayed genetic screens, we systematically studied the biogenesis of a panel of membrane proteins, including several G-protein-coupled receptors (GPCRs). We observed a central role for the insertase, the ER membrane protein complex (EMC), and developed a dual-guide approach to identify genetic modifiers of the EMC. We found that the back of Sec61 (BOS) complex, a component of the multipass translocon, was a physical and genetic interactor of the EMC. Functional and structural analysis of the EMC⋅BOS holocomplex showed that characteristics of a GPCR's soluble domain determine its biogenesis pathway. In contrast to prevailing models, no single insertase handles all substrates. We instead propose a unifying model for coordination between the EMC, the multipass translocon, and Sec61 for the biogenesis of diverse membrane proteins in human cells.

An unexpected journey for BNIP3.

Mitophagy is a cellular process that enables the selective degradation of damaged, dysfunctional, or superfluous mitochondria. During mitophagy, specific proteins recognize and tag mitochondria for degradation. These tagged mitochondria are engulfed by specialized structures called phagophores that then mature into autophagosomes/mitophagosomes. Mitophagosomes subsequently transport their mitochondrial cargo to lysosomes, where the mitochondria are broken down and recycled. While the PINK1-PRKN-dependent mitophagy pathway is well understood, mitophagy can also occur independently of this pathway. BNIP3 and BNIP3L/NIX, paralogous membrane proteins on the outer mitochondrial membrane (OMM), serve as ubiquitin-independent mitophagy receptors. Historically, BNIP3 regulation was thought to be primarily transcriptional through HIF1A (hypoxia inducible factor 1 subunit alpha). However, recent work has revealed a significant post-translational dimension, highlighting the strong role of the ubiquitin-proteasome system (UPS) in BNIP3 regulation. With these emerging concepts in mind, we aimed to develop a unified understanding of how steady-state levels of BNIP3 are established and maintained and how this regulation governs underlying cell physiology.

ER membrane complex (EMC): Structure, functions, and roles in diseases.

The endoplasmic reticulum (ER) is the largest membrane system in eukaryotic cells and is the primary site for the biosynthesis of lipids and carbohydrates, as well as for the folding, assembly, modification, and transport of secreted and integrated membrane proteins. The ER membrane complex (EMC) on the ER membrane is an ER multiprotein complex that affects the quality control of membrane proteins, which is abundant and widely preserved. Its disruption has been found to affect a wide range of processes, including protein and lipid synthesis, organelle communication, endoplasmic reticulum stress, and viral maturation, and may lead to neurodevelopmental disorders and cancer. Therefore, EMC has attracted the attention of many scholars and become a hot field. In this paper, we summarized the main contributions of the research of EMC in the past nearly 15 years, and reviewed the structure and function of EMC as well as its related diseases. We hope this review will promote further progress of research on EMC.

ORMDL3 regulates NLRP3 inflammasome activation by maintaining ER-mitochondria contacts in human macrophages and dictates ulcerative colitis patient outcome.

Genome-wide association studies in inflammatory bowel disease have identified risk loci in the orosomucoid-like protein 3/ORMDL sphingolipid biosynthesis regulator 3 (ORMDL3) gene to confer susceptibility to ulcerative colitis (UC), but the underlying functional relevance remains unexplored. Here, we found that a subpopulation of the UC patients who had higher disease activity shows enhanced expression of ORMDL3 compared to the patients with lower disease activity and the non-UC controls. We also found that the patients showing high ORMDL3 mRNA expression have elevated interleukin-1ß cytokine levels indicating positive correlation. Further, knockdown of ORMDL3 in the human monocyte-derived macrophages resulted in significantly reduced interleukin-1ß release. Mechanistically, we report for the first time that ORMDL3 contributes to a mounting inflammatory response via modulating mitochondrial morphology and activation of the NLRP3 inflammasome. Specifically, we observed an increased fragmentation of mitochondria and enhanced contacts with the endoplasmic reticulum (ER) during ORMDL3 over-expression, enabling efficient NLRP3 inflammasome activation. We show that ORMDL3 that was previously known to be localized in the ER also becomes localized to mitochondria-associated membranes and mitochondria during inflammatory conditions. Additionally, ORMDL3 interacts with mitochondrial dynamic regulating protein Fis-1 present in the mitochondria-associated membrane. Accordingly, knockdown of ORMDL3 in a dextran sodium sulfate -induced colitis mouse model showed reduced colitis severity. Taken together, we have uncovered a functional role for ORMDL3 in mounting inflammation during UC pathogenesis by modulating ER-mitochondrial contact and dynamics.

Visualizing Orientation and Topology of ER Membrane Proteins In Planta.

The orientation of membrane proteins within the lipid bilayer is key to understanding their molecular function. Similarly, the proper topology of multispanning membrane proteins is crucial for their function. Although bioinformatics tools can predict these parameters assessing the presence of hydrophobic protein domains sufficiently long to span the membrane and other structural features, the predictions from different algorithms are often inconsistent. Therefore, experimental analysis becomes mandatory. Redox-based topology analysis exploits the steep gradient in the glutathione redox potential (EGSH) across the ER membrane of about 80 mV to visualize the orientation of ER membrane proteins by fusing the EGSH biosensor roGFP2 to either the N- or the C-termini of the investigated protein sequence. Transient expression of these fusion proteins in tobacco leaves allows direct visualization of orientation and topology of ER membrane proteins in planta. The protocol outlined here is based on either a simple merge of the two excitation channels of roGFP2 or a colocalization analysis of the two channels and thus avoids ratiometric analysis of roGFP2 fluorescence.

SYNJ2BP ameliorates intervertebral disc degeneration by facilitating mitochondria-associated endoplasmic reticulum membrane formation and mitochondrial Zn2+ homeostasis.

Nucleus pulposus (NP) cell function-loss is one main contributor during intervertebral disc degeneration (IDD) progression. Both mitochondria and endoplasmic reticulum (ER) play vital roles in sustaining NP cell homeostasis, while the precise function of ER-mitochondria tethering and cross talk in IDD remain to be clarified. Here, we demonstrated that a notable disruption of mitochondria-associated ER membrane (MAM) was identified in degenerated discs and TBHP-induced NP cells, accompanied by mitochondrial Zn2+ overload and NP cell senescence. Importantly, experimental coupling of MAM contacts by MFN2, a critical regulator of MAM formation, could enhance NLRX1-SLC39A7 complex formation and mitochondrial Zn2+ homeostasis. Further using the sequencing data from TBHP-induced degenerative model of NP cells, combining the reported MAM proteomes, we demonstrated that SYNJ2BP loss was one critical pathological characteristic of NP cell senescence and IDD progression, which showed close relationship with MAM disruption. Overexpression of SYNJ2BP could facilitate MAM contact organization and NLRX1-SLC39A7 complex formation, thus promoted mitochondrial Zn2+ homeostasis, NP cell proliferation and intervertebral disc rejuvenation. Collectively, our present study revealed a critical role of SYNJ2BP in maintaining mitochondrial Zn2+ homeostasis in NP cells during IDD progression, partially via sustaining MAM contact and NLRX1-SLC39A7 complex formation.

Exome sequencing in retinal dystrophy patients reveals a novel candidate gene ER membrane protein complex subunit 3.

Inherited retinal dystrophies (IRDs) are a heterogeneous group of visual disorders caused by different pathogenic mutations in genes and regulatory sequences. The endoplasmic reticulum (ER) membrane protein complex (EMC) subunit 3 (EMC3) is the core unit of the EMC insertase that integrates the transmembrane peptides into lipid bilayers, and the function of its cytoplasmic carboxyl terminus remains to be elucidated. In this study, an insertional mutation c.768insT in the C-terminal coding region of EMC3 was identified and associated with dominant IRDs in a five-generation family. This mutation caused a frameshift in the coding sequence and a gain of an additional 16 amino acid residues (p.L256F-fs-ext21) to form a helix structure in the C-terminus of the EMC3 protein. The mutation is heterozygous with an incomplete penetrance, and cosegregates in all patients examined. This finding indicates that the C-terminus of EMC3 is essential for EMC functions and that EMC3 may be a novel candidate gene for retinal degenerative diseases.

A dual sgRNA library design to probe genetic modifiers using genome-wide CRISPRi screens.

Mapping genetic interactions is essential for determining gene function and defining novel biological pathways. We report a simple to use CRISPR interference (CRISPRi) based platform, compatible with Fluorescence Activated Cell Sorting (FACS)-based reporter screens, to query epistatic relationships at scale. This is enabled by a flexible dual-sgRNA library design that allows for the simultaneous delivery and selection of a fixed sgRNA and a second randomized guide, comprised of a genome-wide library, with a single transduction. We use this approach to identify epistatic relationships for a defined biological pathway, showing both increased sensitivity and specificity than traditional growth screening approaches.

The REEP5/TRAM1 complex binds SARS-CoV-2 NSP3 and promotes virus replication.

IMPORTANCE: Generation of virus-host protein-protein interactions (PPIs) maps may provide clues to uncover SARS-CoV-2-hijacked cellular processes. However, these PPIs maps were created by expressing each viral protein singularly, which does not reflect the life situation in which certain viral proteins synergistically interact with host proteins. Our results reveal the host-viral protein-protein interactome of SARS-CoV-2 NSP3, NSP4, and NSP6 expressed individually or in combination. Furthermore, REEP5/TRAM1 complex interacts with NSP3 at ROs and promotes viral replication. The significance of our research is identifying virus-host interactions that may be targeted for therapeutic intervention.

Mycolactone A vs. B: Multiscale Simulations Reveal the Roles of Localization and Association in Isomer-Specific Toxicity.

Mycolactone is an exotoxin produced by Mycobacterium ulcerans that causes the neglected tropical skin disease Buruli ulcer. This toxin inhibits the Sec61 translocon in the endoplasmic reticulum (ER), preventing the host cell from producing several secretory and transmembrane proteins, resulting in cytotoxic and immunomodulatory effects. Interestingly, only one of the two dominant isoforms of mycolactone is cytotoxic. Here, we investigate the origin of this specificity by performing extensive molecular dynamics (MD) simulations with enhanced free energy sampling to query the association trends of the two isoforms with both the Sec61 translocon, using two distinct cryo-electron microscopy (cryo-EM) models as references, and the ER membrane, which serves as a toxin reservoir prior to association. Our results suggest that mycolactone B (the cytotoxic isoform) has a stronger association with the ER membrane than mycolactone A due to more favorable interactions with membrane lipids and water molecules. This could increase the reservoir of toxin proximal to the Sec61 translocon. In one model of Sec61 inhibited by mycolactone, we find that isomer B interacts more closely with residues thought to play a key role in signal peptide recognition and, thus, are essential for subsequent protein translocation. In the other model, we find that isomer B interacts more closely with the lumenal and lateral gates of the translocon, the dynamics of which are essential for protein translocation. These interactions induce a more closed conformation, which has been suggested to block signal peptide insertion and subsequent protein translocation. Collectively, these findings suggest that isomer B's unique cytotoxicity is a consequence of both increased localization to the ER membrane and channel-locking association with the Sec61 translocon, facets that could be targeted in the development of Buruli Ulcer diagnostics and Sec61-targeted therapeutics.

How host ER membrane chaperones and morphogenic proteins support virus infection.

The multi-functional endoplasmic reticulum (ER) is exploited by viruses to cause infection. Morphologically, this organelle is a highly interconnected membranous network consisting of sheets and tubules whose levels are dynamic, changing in response to cellular conditions. Functionally, the ER is responsible for protein synthesis, folding, secretion and degradation, as well as Ca2+ homeostasis and lipid biosynthesis, with each event catalyzed by defined ER factors. Strikingly, these ER host factors are hijacked by viruses to support different infection steps, including entry, translation, replication, assembly and egress. Although the full repertoire of these ER factors that are hijacked is unknown, recent studies have uncovered several ER membrane machineries that are exploited by viruses - ranging from polyomavirus to flavivirus and coronavirus - to facilitate different steps of their life cycle. These discoveries should provide better understanding of virus infection mechanisms, potentially leading to the development of more effective anti-viral therapies.

Why does the herpes simplex 1 virus-encoded UL49.5 protein fail to inhibit the TAP-dependent antigen presentation?

Herpes simplex virus 1 (HSV-1) is a well-studied herpesvirus that causes various human diseases. Like other herpesviruses, HSV-1 produces the transmembrane glycoprotein N (gN/UL49.5 protein), which has been extensively studied, but its function in HSV-1 remains largely unknown. The amino-acid sequences and lengths of UL49.5 proteins differ between herpesvirus species. It is, therefore, crucial to determine whether and to what extent the spatial structure of UL49.5 orthologs that are transporter associated with antigen processing (TAP) inhibitors (i.e., of bovine herpesvirus 1; BoHV-1) differ from that of non-TAP inhibitors (i.e., of HSV-1). Our study aimed to examine the 3D structure of the HSV-1-encoded UL49.5 protein in an advanced model of the endoplasmic reticulum (ER) membrane using circular dichroism, 2D nuclear magnetic resonance, and multiple-microsecond all-atom molecular dynamics simulations in an ER membrane mimetic environment. According to our findings, the N-terminus of the HSV-1-encoded UL49.5 adopts a highly flexible, unordered structure in the extracellular part due to the presence of a large number of proline and glycine residues. In contrast to the BoHV-1-encoded homolog, the transmembrane region of the HSV-1-encoded UL49.5 is formed by a single long transmembrane α-helix, rather than two helices oriented perpendicularly, while the cytoplasmic part of the protein (C-terminus) has a short unordered structure. Our findings provide valuable experimental structural information on the HSV-1-encoded UL49.5 protein and offer, based on the obtained structure, insight into its lack of biological activity in inhibiting the TAP-dependent antigen presentation pathway.

Depletion of WFS1 compromises mitochondrial function in hiPSC-derived neuronal models of Wolfram syndrome.

Mitochondrial dysfunction involving mitochondria-associated ER membrane (MAM) dysregulation is implicated in the pathogenesis of late-onset neurodegenerative diseases, but understanding is limited for rare early-onset conditions. Loss of the MAM-resident protein WFS1 causes Wolfram syndrome (WS), a rare early-onset neurodegenerative disease that has been linked to mitochondrial abnormalities. Here we demonstrate mitochondrial dysfunction in human induced pluripotent stem cell-derived neuronal cells of WS patients. VDAC1 is identified to interact with WFS1, whereas loss of this interaction in WS cells could compromise mitochondrial function. Restoring WFS1 levels in WS cells reinstates WFS1-VDAC1 interaction, which correlates with an increase in MAMs and mitochondrial network that could positively affect mitochondrial function. Genetic rescue by WFS1 overexpression or pharmacological agents modulating mitochondrial function improves the viability and bioenergetics of WS neurons. Our data implicate a role of WFS1 in regulating mitochondrial functionality and highlight a therapeutic intervention for WS and related rare diseases with mitochondrial defects.

The mitochondrial calcium signaling, regulation, and cellular functions: A novel target for therapeutic medicine in neurological disorders.

Mitochondrial calcium (Ca2+ ) dynamics play critical roles in regulating vital physiological conditions in the brain. Importantly, Mitochondria-associated endoplasmic reticulum (ER) membranes serve different cellular functions including Ca2+ signaling, bioenergetics, phospholipid biosynthesis, cholesterol esterification, programmed cell death, and communication between the two organelles. Several Ca2+ -transport systems specialize at the mitochondria, ER, and their contact sites that provide tight control of mitochondrial Ca2+ signaling at the molecular level. The biological function of Ca2+ channels and transporters as well as the role of mitochondrial Ca2+ signaling in cellular homeostasis can open new perspectives for investigation and molecular intervention. Emerging evidence suggests that abnormalities in ER/mitochondrial brain functions and dysregulation of Ca2+ homeostasis are neuropathological hallmarks of neurological disorders like Alzheimer's disease, but little evidence is available to demonstrate their relationship to disease pathogenesis and therapeutic approaches. In recent years, the detection of the molecular mechanism regulating cellular Ca2+ homeostasis and also mitochondrial functions have expanded the number of targeted treatments. The main experimental data identify beneficial effects, whereas some scientific trials did not meet the expectations. Together with an overview of the important function of mitochondria, this review paper introduced the possible tested therapeutic approaches that target mitochondria in the context of neurodegenerative diseases. Since these treatments in neurological disorders have shown different degrees of progress, it is essential to perform a detailed assessment of the significance of mitochondrial deterioration in neurodegenerative diseases and of a pharmacological treatment at this stage.

The interconnection of endoplasmic reticulum and microtubule and its implication in Hereditary Spastic Paraplegia.

The endoplasmic reticulum (ER) and microtubule (MT) network form extensive contact with each other and their interconnection plays a pivotal role in ER maintenance and distribution as well as MT stability. The ER participates in a variety of biological processes including protein folding and processing, lipid biosynthesis, and Ca2+ storage. MTs specifically regulate cellular architecture, provide routes for transport of molecules or organelles, and mediate signaling events. The ER morphology and dynamics are regulated by a class of ER shaping proteins, which also provide the physical contact structure for linking of ER and MT. In addition to these ER-localized and MT-binding proteins, specific motor proteins and adaptor-linking proteins also mediate bidirectional communication between the two structures. In this review, we summarize the current understanding of the structure and function of ER-MT interconnection. We further highlight the morphologic factors which coordinate the ER-MT network and maintain the normal physiological function of neurons, with their defect causing neurodegenerative diseases such as Hereditary Spastic Paraplegia (HSP). These findings promote our understanding of the pathogenesis of HSP and provide important therapeutic targets for treatment of these diseases.

Ethanol-induced ceramide production causes neuronal apoptosis by increasing MCL-1S-mediated ER-mitochondria contacts.

Heavy alcohol consumption causes neuronal cell death and cognitive impairment. Neuronal cell death induced by ethanol may result from increased production of the sphingolipid metabolite ceramide. However, the molecular mechanisms of neuronal cell death caused by ethanol-induced ceramide production have not been elucidated. Therefore, we investigated the mechanism through which ethanol-induced ceramide production causes neuronal cell apoptosis using human induced-pluripotent stem cell-derived neurons and SH-SY5Y cells and identified the effects of ceramide on memory deficits in C57BL/6 mice. First, we found that ethanol-induced ceramide production was decreased by inhibition of the de novo synthesis pathway, mediated by serine palmitoyltransferase (SPT). The associated alterations of the molecules related to the ceramide pathway suggest that the elevated level of ceramide activated protein phosphatase 1 (PP1), which inhibited the nuclear translocation of serine/arginine-rich splicing factor 1 (SRSF1). This led to aberrant splicing of myeloid cell leukemia 1 (MCL-1) pre-mRNA, which upregulated MCL-1S expression. Our results demonstrated that the interaction of MCL-1S with the inositol 1, 4, 5-trisphosphate receptor (IP3R) increases calcium release from the endoplasmic reticulum (ER) and then activated ER-bound inverted formin 2 (INF2). In addition, we discovered that F-actin polymerization through INF2 activation promoted ER-mitochondria contacts, which induced mitochondrial calcium influx and mitochondrial reactive oxygen species (mtROS) production. Markedly, MCL-1S silencing decreased mitochondria-associated ER membrane (MAM) formation and prevented mitochondrial calcium influx and mtROS accumulation, by inhibiting INF2-dependent actin polymerization interacting with mitochondria. Furthermore, the inhibition of ceramide production in ethanol-fed mice reduced MCL-1S expression, neuronal cell death, and cognitive impairment. In conclusion, we suggest that ethanol-induced ceramide production may lead to mitochondrial calcium overload through MCL-1S-mediated INF2 activation-dependent MAM formation, which promotes neuronal apoptosis.

Mitoregulin controls mitochondrial function and stress-adaptation response during early phase of endoplasmic reticulum stress in breast cancer cells.

The proper regulation of mitochondrial function is important for cellular homeostasis. Especially, in cancer cells, dysregulation of mitochondria is associated with diverse cellular events such as metabolism, redox status, and stress responses. Mitoregulin (MTLN), a micro protein encoded by LINC00116, recently has been reported to control mitochondrial functions in skeletal muscle cells and adipocytes. However, the role of MTLN in cancer cells remains unclear. In the present study, we found that MTLN regulates membrane potential and reactive oxygen species (ROS) generation of mitochondria in breast cancer cells. Moreover, MTLN deficiency resulted in abnormal mitochondria-associated ER membranes (MAMs) formation, which is crucial for stress adaptation. Indeed, the MTLN-deficient breast cancer cells failed to successfully resolve ER (endoplasmic reticulum) stress, and cell vulnerability to ER-stress inducers was significantly enhanced by the downregulation of MTLN. In conclusion, MTLN controls stress-adaptation responses in breast cancer cells as a key regulator of mitochondria-ER harmonization, and thereby its expression level may serve as an indicator of the responsiveness of cancer cells to proteasome inhibitors.

Inhibition of Pyruvate Dehydrogenase Kinase 4 in CD4+ T Cells Ameliorates Intestinal Inflammation.

BACKGROUND & AIMS: Despite recent evidence supporting the metabolic plasticity of CD4+ T cells, it is uncertain whether the metabolic checkpoint pyruvate dehydrogenase kinase (PDK) in T cells plays a role in the pathogenesis of colitis. METHODS: To investigate the role of PDK4 in colitis, we used dextran sulfate sodium (DSS)-induced colitis and T-cell transfer colitis models based on mice with constitutive knockout (KO) or CD4+ T-cell-specific KO of PDK4 (Pdk4fl/flCD4Cre). The effect of PDK4 deletion on T-cell activation was also studied in vitro. Furthermore, we examined the effects of a pharmacologic inhibitor of PDK4 on colitis. RESULTS: Expression of PDK4 increased during colitis development in a DSS-induced colitis model. Phosphorylated PDHE1α, a substrate of PDK4, accumulated in CD4+ T cells in the lamina propria of patients with inflammatory bowel disease. Both constitutive KO and CD4+ T-cell-specific deletion of PDK4 delayed DSS-induced colitis. Adoptive transfer of PDK4-deficient CD4+ T cells attenuated murine colitis, and PDK4 deficiency resulted in decreased activation of CD4+ T cells and attenuated aerobic glycolysis. Mechanistically, there were fewer endoplasmic reticulum-mitochondria contact sites, which are responsible for interorganelle calcium transfer, in PDK4-deficient CD4+ T cells. Consistent with this, GM-10395, a novel inhibitor of PDK4, suppressed T-cell activation by reducing endoplasmic reticulum-mitochondria calcium transfer, thereby ameliorating murine colitis. CONCLUSIONS: PDK4 deletion from CD4+ T cells mitigates colitis by metabolic and calcium signaling modulation, suggesting PDK4 as a potential therapeutic target for IBD.

Liver mitochondria-associated endoplasmic reticulum membrane proteomics for studying the effects of ZiBuPiYin recipe on Zucker diabetic fatty rats after chronic psychological stress.

Type 2 diabetes mellitus (T2DM) is a complex metabolic disease with multiple etiologies, involving both genetic and environmental factors. With changes associated with modern life, increasing attention has been paid to chronic psychological stressors such as work stress. Chronic psychological stress can induce or aggravate diabetes mellitus, and conversely, with the deterioration of T2DM, patients often experience different degrees of depression, anxiety, and other negative emotions. In order to clarify the role of ZiBuPiYin recipe (ZBPYR) in regulating the liver mitochondria-associated endoplasmic reticulum membrane proteome to improve T2DM with chronic psychological stress, differentially expressed proteins (DEPs) were identified among Zucker lean littermates (control group), chronic psychological stress T2DM rats (model group), and ZBPYR administration rats (ZBPYR group) through iTRAQ with LC-MS/MS. Using Mfuzz soft clustering analysis, DEPs were divided into six different clusters. Clusters 1-6 contained 5, 68, 44, 57, 28, and 32 DEPs, respectively. Given that ZBPYR can alleviate T2DM symptoms and affect exploratory behavior during T2DM with chronic psychological stress, we focused on the clusters with opposite expression trends between model:control and ZBPYR:model groups. We screened out the DEPs in clusters 1, 3, and 4, which may be good candidates for the prevention and treatment of T2DM with chronic psychological stress, and further conducted bioinformatics analyses. DEPs were mainly involved in the insulin signaling pathway, oxidative phosphorylation, tricarboxylic acid cycle, amino acid metabolism, lysosome-related processes, and lipid metabolism. This may indicate the pathogenic basis of T2DM with chronic psychological stress and the potential therapeutic mechanism of ZBPYR. In addition, two key proteins, lysosome-associated protein (Lamp2) and tricarboxylic acid cycle-related protein (Suclg1), may represent novel biomarkers for T2DM with chronic psychological stress and drug targets of ZBPYR. Western blot analyses also showed similar expression patterns of these two proteins in liver MAMs of the model and ZBPYR groups.