Quantitative Proteomic Analysis Research Articles

Ropivacaine inhibits the malignant behavior of lung cancer cells by regulating retinoblastoma-binding protein 4.

Ropivacaine is a local anesthetic commonly used in regional nerve blocks to manage perioperative pain during lung cancer surgery. Recently, the antitumor potential of ropivacaine has received considerable attention. Our previous study showed that ropivacaine treatment inhibits the malignant behavior of lung cancer cells in vitro. However, the potential targets of ropivacaine in lung cancer cells have not yet been fully identified. This study aimed to explore the antitumor effects and mechanisms of action of ropivacaine in lung cancer. Lung cancer A549 cells were treated with or without 1 mM ropivacaine for 48 h. Quantitative proteomics was performed to identify the differentially expressed proteins (DEPs) triggered by ropivacaine treatment. STRING and Cytoscape were used to construct protein-protein interaction (PPI) networks and analyze the most significant hub genes. Overexpression plasmids and small interfering RNA were used to modulate the expression of key DEPs in A549 and H1299 cells. MTS, transwell assays, and flow cytometry were performed to determine whether the key DEPs were closely related to the anticancer effect of ropivacaine on the malignant behavior of A549 and H1299 cells. Quantitative proteomic analysis identified 327 DEPs (185 upregulated and 142 downregulated proteins) following ropivacaine treatment. Retinoblastoma-binding protein 4 (RBBP4) was one of the downregulated DEPs and was selected as the hub protein. TCGA database showed that RBBP4 was significantly upregulated in lung cancer and was associated with poor patient prognosis. Inhibition of RBBP4 by siRNA resulted in a significant decrease in the proliferation and invasive capacity of lung cancer cells and the induction of cell cycle arrest. Additionally, the results indicated RBBP4 knockdown enhanced antitumor effect of ropivacaine on A549 and H1299 cells. Conversely, the overexpression of RBBP4 using plasmids reversed the inhibitory effects of ropivacaine. Our data suggest that ropivacaine suppresses lung cancer cell malignancy by downregulating RBBP4 protein expression, which may help clarify the mechanisms underlying the antitumor effects of ropivacaine.

ITRAQ-based proteomics analysis of human ectopic endometrial stromal cells treated by Maqian essential oil

BackgroundEndometriosis is a common and complex syndrome characterized by the presence of endometrial-like tissue outside the uterus. Chinese medicine has been recently found to show good efficacy in treating endometriosis. Our previous results revealed that Maqian fruit essential oil (MQEO) could inhibit the proliferation and induce apoptosis of ectopic endometrial stromal cells (EESCs), but the mechanisms remain unclear. In this study, we aim to explore the molecular mechanism of MQEO’s specific effects in EESCs.MethodsWe conducted a quantitative proteomics analysis by iTRAQ on EESCs treated with MQEO or DMSO. Then deep analysis was performed based on differentially expressed proteins, including Gene Ontology enrichment analysis, pathway enrichment analysis and protein interaction analysis. Candidate protein targets were subsequently verified by western blotting.ResultsAmong 6575 identified proteins, 435 proteins exhibited altered expression levels in MQEO-treated EESCs. Of these proteins, most were distributed in signal transduction as well as immune system and the most significantly altered pathway was complement and coagulation cascades. Moreover, two differentially expressed proteins (Heme oxygenase 1 and Acyl-CoA 6-desaturase) were verified and they can be potential biomarkers for endometriosis treatment.ConclusionsOur proteomic analysis revealed distinct protein expression patterns induced by MQEO treatment in EESCs, highlighting the potential of MQEO for endometriosis treatment and biomarker discovery.

TMT quantitative proteomics reveals key proteins relevant to microRNA-1-mediated regulation in osteoarthritis

Osteoarthritis (OA) is the second-commonest arthritis, but pathogenic and regulatory mechanisms underlying OA remain incompletely understood. Here, we aimed to identify the mechanisms associated with microRNA-1 (miR-1) treatment of OA in rodent OA models using a proteomic approach. First, N = 18 Sprague Dawley (SD) rats underwent sham surgery (n = 6) or ACL transection (n = 12), followed at an interval of one week by randomization of the ACL transection group to intra-articular administration of either 50 µL placebo (control group) or miR-1 agomir, a mimic of endogenous miR-1 (experimental group). After allowing for eight weeks of remodeling, articular cartilage tissue was harvested and immunohistochemically stained for the presence of MMP-13. Second, N = 30 Col2a1-cre-ERT2 /GFPf1/fl -RFP-miR-1 transgenic mice were randomized to intra-articular administration of either placebo (control group, N = 15) or tamoxifen, an inducer of miR-1 expression (experimental group, N = 15), before undergoing surgical disruption of the medial meniscus (DMM) after an interval of five days. After allowing for eight weeks of remodeling, articular cartilage tissue was harvested and underwent differential proteomic analysis. Specifically, tandem mass tagging (TMT) quantitative proteomic analysis was employed to identify inter-group differentially-expressed proteins (DEP), and selected DEPs were validated using real-time quantitative polymerase chain reaction (RT-qPCR) technology. Immunohistochemically-detected MMP-13 expression was significantly lower in the experimental rat group, and proteomic analyses of mouse tissue homogenate demonstrated that of 3526 identified proteins, 345 were differentially expressed (relative up- and down-regulation) in the experimental group. Proteins Fn1, P4ha1, P4ha2, Acan, F2, Col3a1, Fga, Rps29, Rpl34, and Fgg were the *top ten most-connected proteins, implying that miR-1 may regulate an expression network involving these proteins. Of these ten proteins, three were selected for further validation by RT-qPCR: the transcript of Fn1, known to be associated with OA, exhibited relative upregulation in the experimental group, whereas the transcripts of P4ha1 and Acan exhibited relative downregulation. These proteins may thus represent key miR-1 targets during OA-regulatory mechanisms, and may provide additional insights regarding therapeutic mechanisms of miR-1 in context of OA.

Identification of a Candidate Gene Regulating Intramuscular Fat Content in Pigs through the Integrative Analysis of Transcriptomics and Proteomics Data.

Pork is a widely consumed source of animal protein worldwide, and the intramuscular fat (IMF) content in pork plays a crucial role in determining its quality. In this study, we sought to identify candidate genes that regulate IMF deposition in pigs. We performed tandem mass tags (TMT)-based quantitative proteomics analysis using Longissimus dorsi (LD) muscle samples obtained from eight pigs with extremely high and low IMF content among a group of 28 Duroc pigs and identified 50 differentially abundant proteins (DAPs). Additionally, we compared the proteomics data with RNA-sequencing data obtained in our previous study and identified TUSC5 as a differentially expressed gene corresponding to the relevant DAP. To investigate the potential role of TUSC5 in adipogenesis, we suppressed TUSC5 expression in mouse 3T3-L1 preadipocytes using short hairpin RNA (shRNA) and observed a significant reduction in the differentiation of 3T3-L1 cells into adipocytes, as indicated by Oil Red O staining and triglyceride content. Moreover, we observed a reduction in the expression of genes associated with adipogenesis (PPARG, CEBPA, FABP4, and FASN) following TUSC5 suppression. Through an integrative analysis of transcriptomics and proteomics data, our study identified TUSC5 as a crucial candidate gene associated with the regulation of IMF content in pigs.

Microplastics exposure induced and exacerbated the development of systemic lupus erythematosus in mice

Environmental exposure may function as a contributing risk factor in the development of systemic lupus erythematosus (SLE). Recently, the global issue of microplastics (MPs) pollution has garnered increasing concern, yet its potential impact on SLE remains unexplored. This study seeks to elucidate the ramifications of MPs exposure on lupus manifestations in spontaneous lupus MRL/lpr mice and normal C57L/6 mice. MPs exposure demonstrated the capacity to induce lupus-like symptoms in C57BL/6 mice and exacerbate lupus symptoms in MRL/lpr mice. This was manifested by MPs triggering abnormal elevation of spleen DN T, plasma cells, serum anti-dsDNA, ANA, IL-6, and TNF-α, coupled with a reduction in spleen CD4+/CD8+ cell ratio, and impairment in renal pathology. Moreover, a 4D-DIA quantitative proteomic analysis was employed to unveil substantial alterations in renal proteins attributed to MPs exposure. The findings indicated that the KEGG pathways significantly enriched by MPs-associated different proteins in C57BL/6 mice were closely aligned with the enriched KEGG pathways associated with lupus. Unlike C57BL/6 mice, there were no significantly enriched KEGG pathways identified among the MPs-associated different proteins in MRL/lpr mice. In addition, proteins related to the SLE pathway illuminated that MPs exposure induced renal damage through activation of MHCII and histone H3, culminating in the production of MAC in both C57BL/6 and MRL/lpr mice. However, a specific elevation in cathepsin and elastase caused by MPs was observed in C57BL/6 mice but not in MRL/lpr mice. This study represents a significant stride in bridging the existing knowledge gap pertaining to the intricate relationship between MPs exposure and the development of SLE.

Exploration of the pathophysiology of high myopia via proteomic profiling of human corneal stromal lenticules

This study aimed to investigate the underlying pathophysiology of high myopia by analyzing the proteome of human corneal stromal lenticule samples obtained through small incision lenticule extraction (SMILE). A total of thirty-two patients who underwent SMILE were included in the study. Label-free quantitative proteomic analysis was performed on corneal stromal lenticule samples, equally representing high myopia (n = 10) and low myopia (n = 10) groups. The identified and profiled lenticule proteomes were analyzed using in silico tools to explore biological characteristics of differentially expressed proteins (DEPs). Additionally, LASSO regression and random forest model were employed to identify key proteins associated with the pathophysiology of high myopia. The DEPs were found to be closely linked to immune activation, extracellular matrix, and cell adhesion-related pathways according to gene ontology analysis. Specifically, decreased expression of COL1A1 and increased expression of CDH11 were associated with the pathogenesis of high myopia and validated by western blotting (n = 6) and quantitative real time polymerase chain reaction (n = 6). Overall, this study provides evidence that COL1A1 and CDH11 may contribute to the pathophysiology of high myopia based on comparative proteomic profiling of human corneal stromal lenticules obtained through SMILE.

Type II metacaspase mediates light-dependent programmed cell death in Chlamydomonas reinhardtii.

Among the crucial processes that preside over the destiny of cells from any type of organism are those involving their self-destruction. This process is well characterized and conceptually logical to understand in multicellular organisms; however, the levels of knowledge and comprehension of its existence are still quite enigmatic in unicellular organisms. We use Chlamydomonas (Chlamydomonas reinhardtii) to lay the foundation for understanding the mechanisms of programmed cell death (PCD) in a unicellular photosynthetic organism. In this paper, we show that while PCD induces the death of a proportion of cells, it allows the survival of the remaining population. A quantitative proteomic analysis aiming at unveiling the proteome of PCD in Chlamydomonas allowed us to identify key proteins that led to the discovery of essential mechanisms. We show that in Chlamydomonas, PCD relies on the light dependence of a photosynthetic organism to generate reactive oxygen species and induce cell death. Finally, we obtained and characterized mutants for the 2 metacaspase genes in Chlamydomonas and showed that a type II metacaspase is essential for PCD execution.

Effects of Doxorubicin on Extracellular Matrix Regulation in Primary Cardiac Fibroblasts from Mice

ObjectiveDoxorubicin (DOX) is a highly effective chemotherapeutic used to treat many adult and pediatric cancers. However, its use is limited due to a dose-dependent cardiotoxicity, which can lead to lethal cardiomyopathy. In contrast to the extensive research efforts on toxic effects of DOX in cardiomyocytes, its effects and mechanisms on cardiac extracellular matrix (ECM) homeostasis and remodeling are poorly understood. In this study, we examined the potential effects of DOX on cardiac ECM to further our mechanistic understanding of DOX-induced cardiotoxicity.ResultsDOX-induced significant down-regulation of several ECM related genes in primary cardiac fibroblasts, including Adamts1, Adamts5, Col4a1, Col4a2, Col5a1, Fbln1, Lama2, Mmp11, Mmp14, Postn, and TGFβ. Quantitative proteomics analysis revealed significant global changes in the fibroblast proteome following DOX treatment. A pathway analysis using iPathwayGuide of the differentially expressed proteins revealed changes in a list of biological pathways that involve cell adhesion, cytotoxicity, and inflammation. An apparent increase in Picrosirius red staining indicated that DOX-induced an increase in collagen production in cardiac primary fibroblasts after 3-day treatment. No significant changes in collagen organization nor glycoprotein production were observed.

ITRAQ-based quantitative proteomic analysis of the antibacterial mechanism of silver nanoparticles against multidrug-resistant Streptococcus suis.

The increase in antibiotic resistance of bacteria has become a major concern in clinical treatment. Silver nanoparticles (AgNPs) have significant antibacterial effects against Streptococcus suis. Therefore, this study aimed to investigate the antibacterial activity and mechanism of action of AgNPs against multidrug-resistant S. suis. The effect of AgNPs on the morphology of multidrug-resistant S. suis was observed using scanning electron microscopy (SEM). Differentially expressed proteins were analyzed by iTRAQ quantitative proteomics, and the production of reactive oxygen species (ROS) was assayed by H2DCF-DA staining. SEM showed that AgNPs disrupted the normal morphology of multidrug-resistant S. suis and the integrity of the biofilm structure. Quantitative proteomic analysis revealed that a large number of cell wall synthesis-related proteins, such as penicillin-binding protein and some cell cycle proteins, such as the cell division protein FtsZ and chromosomal replication initiator protein DnaA, were downregulated after treatment with 25 μg/mL AgNPs. Significant changes were also observed in the expression of the antioxidant enzymes glutathione reductase, alkyl hydroperoxides-like protein, α/β superfamily hydrolases/acyltransferases, and glutathione disulfide reductases. ROS production in S. suis positively correlated with AgNP concentration. The potential antibacterial mechanism of AgNPs may involve disrupting the normal morphology of bacteria by inhibiting the synthesis of cell wall peptidoglycans and inhibiting the growth of bacteria by inhibiting the cell division protein FtsZ and Chromosomal replication initiator protein DnaA. High oxidative stress may be a significant cause of bacterial death. The potential mechanism by which AgNPs inhibit S. suis biofilm formation may involve affecting bacterial adhesion and interfering with the quorum sensing system.

Chicken egg white precipitates induced by water dilution

Chicken egg whites (whole egg white, EW; thick egg white, TKEW; thin egg white, TNEW) become turbid and are accompanied by the formation of precipitates after being diluted with an equal mass of deionized water. The precipitates of TKEW induced by water dilution (Thick Egg White Precipitates, TKEWP) account for 14.47 % of TKEW total dry matter, much higher than that of thin egg white precipitates (TNEWP) (1.51 %) and whole egg white precipitates (EWP) (5.53 %). Quantitative proteomic analysis identified 27 differentially abundant proteins (p < 0.05) among EW, EWP, TNEWP, and TKEWP. Lysozyme was found to be a key protein in the formation of EW precipitates induced by water dilution, as its abundance was significantly higher in TNEWP and TKEWP. Mucin-5B (α-ovomucin) had the highest abundance in TKEWP, suggesting that its insolubility is one of the important factors contributing to the large formation of TKEWP. This paper systematically studies the formation, characteristics, and composition of egg white precipitation caused by water dilution, and puts forward a new understanding of the processing characteristics of egg white liquid, thus laying a theoretical foundation for further research methods to reduce egg white precipitation by water dilution.

Potential prognostic biomarkers of hepatocellular carcinoma based on 4D label-free quantitative proteomics analysis pilot investigation.

Hepatocellular carcinoma carries a poor prognosis and poses a serious threat to global health. Currently, there are few potential prognostic biomarkers available for the prognosis of hepatocellular carcinoma. This pilot study used 4D label-free quantitative proteomics to compare the proteomes of hepatocellular carcinoma and adjacent non-tumor tissue. A total of 66,075 peptides, 6363 identified proteins, and 772 differentially expressed proteins were identified in specimens from three hepatocellular carcinoma patients. Through functional enrichment analysis of differentially expressed proteins by Gene Ontology, KEGG pathway, and protein domain, we identified proteins with similar functions. Twelve differentially expressed proteins (RPL17, RPL27, RPL27A, RPS5, RPS16, RSL1D1, DDX18, RRP12, TARS2, YARS2, MARS2, and NARS1) were selected for identification and validation by parallel reaction monitoring. Subsequent Western blotting confirmed overexpression of RPL27, RPS16, and TARS2 in hepatocellular carcinoma compared to non-tumor tissue in 16 pairs of clinical samples. Analysis of The Cancer Genome Atlas datasets associated the increased expression of these proteins with poor prognosis. Tissue microarray revealed a negative association between high expression of RPL27 and TARS2 and the prognosis of hepatocellular carcinoma patients, although RPS16 was not significant. These data suggest that RPL27 and TARS2 play an important role in hepatocellular carcinoma progression and may be potential prognostic biomarkers of overall survival in hepatocellular carcinoma patients.

Lamin A/C mediates microglial activation by modulating cell proliferation and immune response.

Lamin A/C is involved in macrophage activation and premature aging, also known as progeria. As the resident macrophage in brain, overactivation of microglia causes brain inflammation, promoting aging and brain disease. In this study, we investigated the role of Lamin A/C in microglial activation and its impact on progeria using Lmna-/- mice, primary microglia, Lmna knockout (Lmna-KO) and Lmna-knockdown (Lmna-KD) BV2 cell lines. We found that the microglial activation signatures, including cell proliferation, morphology changes, and proinflammatory cytokine secretion (IL-1β, IL-6, and TNF-α), were significantly suppressed in all Lamin A/C-deficient models when stimulated with LPS. TMT-based quantitative proteomic and bioinformatic analysis were further applied to explore the mechanism of Lamin A/C-regulated microglia activation from the proteome level. The results revealed that immune response and phagocytosis were impaired in Lmna-/- microglia. Stat1 was identified as the hub protein in the mechanism by which Lamin A/C regulates microglial activation. Additionally, DNA replication, chromatin organization, and mRNA processing were also altered by Lamin A/C, with Ki67 fulfilling the main hub function. Lamin A/C is a mechanosensitive protein and, the immune- and proliferation-related biological processes are also regulated by mechanotransduction. We speculate that Lamin A/C-mediated mechanotransduction is required for microglial activation. Our study proposes a novel mechanism for microglial activation mediated by Lamin A/C.

Developing a Toolbox of Antibodies Validated for Array Tomography-Based Imaging of Brain Synapses.

Antibody (Ab)-based imaging techniques rely on reagents whose performance may be application specific. Because commercial antibodies are validated for only a few purposes, users interested in other applications may have to perform extensive in-house antibody testing. Here, we present a novel application-specific proxy screening step to efficiently identify candidate antibodies for array tomography (AT), a serial section volume microscopy technique for high-dimensional quantitative analysis of the cellular proteome. To identify antibodies suitable for AT-based analysis of synapses in mammalian brain, we introduce a heterologous cell-based assay that simulates characteristic features of AT, such as chemical fixation and resin embedding that are likely to influence antibody binding. The assay was included into an initial screening strategy to generate monoclonal antibodies that can be used for AT. This approach simplifies the screening of candidate antibodies and has high predictive value for identifying antibodies suitable for AT analyses. In addition, we have created a comprehensive database of AT-validated antibodies with a neuroscience focus and show that these antibodies have a high likelihood of success for postembedding applications in general, including immunogold electron microscopy. The generation of a large and growing toolbox of AT-compatible antibodies will further enhance the value of this imaging technique.

Mitochondrial and contractile dysfunction in a swine model of early chronic kidney disease

Abstract Background Numerous clinical trials have demonstrated that patients with kidney damage have an increased chance of developing heart failure, and the risk is compounded in people with diabetes. Cardiac remodeling occurs at very early stages of kidney injury but studies undertaken to pinpoint the common biomarkers of cardiorenal signaling are still inadequate, which may be due to the fact that research done on rodent models does not fully reflect the complexity of the disease. We and our collaborators established swine models for diabetes (DM- insulin gene INS mutation) and chronic kidney disease (CKD- renal microembolisation) to overcome the translational gap associated with rodent models. Purpose In order to provide mechanistic as well as therapeutic insights, we used proteomic analyses to elucidate the pathophysiological effects of DM and CKD on the heart. Methods We used microspheres to embolise both kidneys of wild type and diabetic swine at 10-12 weeks of age and 6 months later we performed quantitative LC-MS-MS based proteomic analyses of left ventricular tissue from these four groups (wild type (WT), wild type with CKD (WT_CKD), diabetic (DM) and diabetic with CKD (DM_CKD). STRING bioinformatics software was used to identify significantly enriched pathways. Results Masson Trichrome staining confirmed the presence of kidney damage and fibrosis in CKD animals (Fig1 A). Quantitative proteomic analysis detected significant differences in the left ventricle of the WT or DM swine when compared to those with CKD using a volcano plot (Fig1B,C). STRING pre-ranked functional analysis showed pathway enrichments in both the comparisons (Fig2A,B). We found that proteins involved in the remodeling of the extracellular matrix (ECM), such as C3, F9, Col6A2, and FBN1, were downregulated in both groups (WT CKD and DM CKD as compared to WT and DM respectively). Interestingly, in WT_CKD we found that mitochondrial proteins like cytochrome oxidase (MT-CO2) and ATP5 synthase subunit were downregulated suggesting mitochondrial dysfunction. Strong indicators of this dysfunction include pathways with altered regulation of reactive oxygen species like hydrogen peroxide and substantial downregulation of contractile proteins like phospholamban, which maintains the Ca2+ homeostasis. DM_CKD on the other hand when compared to DM suggested presence of an inflammatory milieu with alterations of metabolic, wound healing and immune cell regulation pathways. Conclusions Our proteomic data in swine model provides strong evidence that mild CKD can induce early alterations in contractile proteins of the heart due to mitochondrial dysfunction along with ECM remodeling. In the presence of diabetes, metabolic and immune cells play a more predominant role in early part of the disease. Thus, we aim to further our understanding by applying our proteomic findings in conjunction with functional hemodynamic data in this swine model of cardiorenal disease.Figure 1Figure 2

Quantitative transcriptomic and proteomic analysis reveals corosolic acid inhibiting bladder cancer via suppressing cell cycle and inducing mitophagy in vitro and in vivo

Corosolic acid (CA) is a plant-derived terpenoid compound with many health benefits. However, the anti-tumor effects of CA in bladder cancer remain unexplored. Here, we found that CA inhibited bladder tumor both in vitro and in vivo, and had no significant toxicity in mice. With the aid of transcriptomics and proteomics, we elucidated the regulatory network mechanism of CA inhibiting bladder cancer. Through cell viability detection, cell fluorescence staining and flow cytometry, we discovered that CA inhibited bladder cancer mainly through blocking cell cycle. Interestingly, CA played anticancer roles by distinct mechanisms at different concentrations: low concentrations (<7.0 μg/ml) of CA mainly inhibited DNA synthesis by downregulating TOP2A and LIG1, and diminished mitosis by downregulating CCNA2, CCNB1, CDC20, and RRM2; high concentrations (≥7.0 μg/ml) of CA induced cell death through triggering mitophagy via upregulating NBR1, TAXBP1, SQSTM1/P62, and UBB. CA, as a natural molecule of homology of medicine and food, is of great significance for the prevention and treatment of cancer patients following clarifying its anti-cancer mechanism. This study provides a comprehensive understanding of the pharmacological mechanism of CA inhibition in bladder cancer, which is helpful for the development of new anti-tumor drugs based on CA.

Neuronal survival factor TAFA2 suppresses apoptosis through binding to ADGRL1 and activating cAMP/PKA/CREB/BCL2 signaling pathway

AimsTAFA2, a cytokine specifically expressed in the central nervous system, plays a vital role in neuronal cell survival. TAFA2 deficiency has been correlated to various neurological disorders in mice and humans. However, the underlying mechanism remains elusive, especially its membrane-binding receptor through which TAFA2 functions. This study aimed to identify the specific binding receptor responsible for the anti-apoptotic effects of TAFA2. Main methodCo-immunoprecipitation (Co-IP) and quantitative mass spectrometry-based proteomic analysis were employed to identify potential TAFA2 binding proteins in V5 knockin mouse brain lysates. Subsequent validation involved in vitro and in vivo Co-IP and pull-down using specific antibodies. The functional analysis included evaluating the effects of ADGRL1 knockout, overexpression, and Lectin-like domain (Lec) deletion mutant on TAFA2's anti-apoptotic activity and analyzing the intracellular signaling pathways mediated by TAFA2 through ADGRL1. Key findingsOur study identified ADGRL1 as a potential receptor for TAFA2, which directly binds to TAFA2 through its lectin-like domain. Overexpression ADGRL1, but not ADGRL1ΔLec, induced apoptosis, which could be effectively suppressed by recombinant TAFA2 (rTAFA2). In ADGRL1−/− cells or re-introducing with ADGRL1ΔLec, responses to rTAFA2 in suppressing cell apoptosis were compromised. Increased cAMP, p-PKA, p-CREB, and BCL2 levels were also observed in response to rTAFA2 treatment, with these responses attenuated in ADGRL1−/− or ADGRL1ΔLec-expressing cells. SignificanceOur results demonstrated that TAFA2 directly binds to the lectin-like domain of ADGRL1, activating cAMP/PKA/CREB/BCL2 signaling pathway, which is crucial in preventing cell death. These results implicate TAFA2 and its receptor ADGRL1 as potential therapeutic targets for neurological disorders.

HIV-1 Nef Changes the Proteome of T Cells Extracellular Vesicles Depleting IFITMs and Other Antiviral Factors

Extracellular vesicles (EVs) are biomolecule carriers for intercellular communication in health and disease. Nef is a HIV virulence factor that is released from cells within EVs and is present in plasma EVs of HIV-1 infected individuals. We performed a quantitative proteomic analysis to fully characterize the Nef-induced changes in protein composition of T cell-derived EVs and identify novel host targets of HIV. Several proteins with well-described roles in infection or not previously associated with HIV pathogenesis were specifically modulated by Nef in EVs. Among the downregulated proteins are the interferon-induced transmembrane 1, 2, and 3 (IFITM1-3) proteins, broad-spectrum antiviral factors known to be cell-to-cell transferable by EVs. We demonstrate that Nef depletes IFITM1-3 from EVs by excluding these proteins from the plasma membrane and lipid rafts, which are sites of EVs biogenesis in T cells. Our data establish Nef as a modulator of EVs' global protein content and as an HIV factor that antagonizes IFITMs.

Targeting NADPH Oxidase and Integrin α5β1 to Inhibit Neutrophil Extracellular Traps-Mediated Metastasis in Colorectal Cancer.

Metastasis leads to a high mortality rate in colorectal cancer (CRC). Increased neutrophil extracellular traps (NETs) formation is one of the main causes of metastasis. However, the mechanism of NETs-mediated metastasis remains unclear and effective treatments are lacking. In this study, we found neutrophils from CRC patients have enhanced NETs formation capacity and increased NETs positively correlate with CRC progression. By quantitative proteomic analysis of clinical samples and cell lines, we found that decreased secreted protein acidic and rich in cysteine (SPARC) results in massive NETs formation and integrin α5β1 is the hub protein of NETs-tumor cell interaction. Mechanistically, SPARC regulates the activation of the nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) pathway by interacting with the receptor for activated C kinase 1 (RACK1). Over-activated NADPH oxidase generates more reactive oxygen species (ROS), leading to the release of NETs. Then, NETs upregulate the expression of integrin α5β1 in tumor cells, which enhances adhesion and activates the downstream signaling pathways to promote proliferation and migration. The combination of NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI) and integrin α5β1 inhibitor ATN-161 (Ac-PHSCN-NH2) effectively suppresses tumor progression in vivo. Our work reveals the mechanistic link between NETs and tumor progression and suggests a combination therapy against NETs-mediated metastasis for CRC.

Shenge Formula attenuates high-fat diet-induced obesity and fatty liver via inhibiting ACOX1

BackgroundNonalcoholic fatty liver disease (NAFLD) is the leading cause of chronic liver disease worldwide. Shenge Formula (SGF) is a traditional Chinese medicine that has been used in the clinical treatment of NAFLD, and its therapeutic potential in patients and NAFLD animal models has been demonstrated in numerous studies. However, its underlying mechanism for treating NAFLD remains unclear. PurposeThe aim of this study was to investigate the mechanism of SGF in the treatment of NAFLD using the proteomics strategy. MethodsUltra-high performance liquid chromatography-mass spectrometry (UPLC-MS) was used to determine the main components of SGF. A mouse model of nonalcoholic fatty liver disease was constructed by feeding mice with a high-fat diet for 16 weeks. SGF was administered for an additional 8 weeks, and metformin was used as a positive control. Liver sections were subjected to histopathological assessments. LC-MS/MS was used for the label-free quantitative proteomic analysis of liver tissues. Candidate proteins and pathways were validated both in vivo and in vitro through qRT-PCR, western blot, and immunohistochemistry. The functions of the validated pathways were further investigated using the inhibition strategy. ResultsThirty-nine ingredients were identified in SGF extracts, which were considered to be key compounds in the treatment of NAFLD. SGF administration attenuated obesity and fatty liver by reducing the body weight and liver weight in HFD-fed mice. It also relieved HFD-induced insulin resistance. More importantly, hepatic steatosis was significantly attenuated by SGF administration both in vivo and in vitro. Proteomic profiling of mouse liver tissues identified 184 differential expressed proteins (DEPs) associated with SGF treatment. Bioinformatic analysis of DEPs revealed that regulating the lipid metabolism and energy consumption process of hepatocytes was the main role of SGF in NAFLD treatment. This also indicated that ACOX1 might be the potential target of SGF, which was subsequently verified both in vitro and in vivo. The results demonstrated that SGF inhibited ACOX1 activity, thereby activating PPARα and upregulating CPT1A expression. Increased CPT1A expression promoted mitochondrial β-oxidation, leading to reduced lipid accumulation in hepatocytes. ConclusionsOverall, our findings confirmed the protective effect of SGF against NAFLD and revealed the underlying molecular mechanism of regulating lipid metabolism.

UBC9 Deficiency Accelerates AML Progression By Downregulating PARP1 Sumoylation and Subsequently Activating the NF-ΚB Pathway

Acute myeloid leukemia (AML) is the most common acute leukemia in adults and is a disease of heterogeneous clonal hematopoietic progenitor cells. Despite the relentless development of therapeutic options, including chemotherapy, and identification of many new therapeutic targets, the prognosis of AML is still poor, with a 5-year survival rate of approximately 35% in patients younger than 60 years old and 10% in patients older than 60 years, which may be due to the high degree of AML heterogeneity. Targeting the shared mechanisms of action among subclones has emerged led to find novel strategies, such as targeting protein post post-translational modification. Here we show that the impact of the UBC9 (the only E2 conjugation enzyme) and its related SUMOylation, a critical and reversible posttranslational modification as ubiquitination, on AML progression, to provide novel and potent sight for AML therapy. To identify the role of UBC9 expression in the clinical outcome of AML patients, we measured the expression of key SUMOylation genes, including SAE1, UBA2, UBC9, SUMO1, SUMO2, SUMO3, PIAS1, PIAS2, PIAS3, PIAS4, CBX4, and RANBP2, in the TCGA dataset (n=164) by using a multivariate survival curve based on the online tool SurvExpress. Our results showed that the survival probability curves of the low- and high-risk groups were significantly different due to the differences in the levels of expression of key SUMOylation genes ( p = 0.0011), and we found that the expression level of UBC9 was lower in the high-risk group than in the low-risk group. We next analyzed the UBC9 expression level and the prognosis of AML patients from the Therapeutically Applicable Research To Generate Effective Treatment dataset (n=156), which showed that AML patients with low UBC9 expression showed shorter overall survival ( p = 0.018)(Figure 1). In addition, real-time PCR showed that the UBC9 expression level in bone marrow samples from patients with de novo AML was significantly downregulated in both the high-risk patients based on National Comprehensive Cancer Network guidelines and the liver or spleen enlargement patients ( p = 0.01 and p = 0.001, respectively) (Figure 1). We used RNA interference system to knock out Ubc9 in a mouse model of MLL-AF9 and MLL-ENL induced AML. We observed the survival of AML mice and found that Ubc9 fl/+;Vav-Cre recipient mice showed a significantly shorter survival time than Ubc9 fl/+ mice (Figure 2). In addition, apoptosis experiments revealed reduced apoptosis of leukemia cells and leukemia stem cells in Ubc9 fl/+;Vav-cre mice. HE staining showed significantly higher leukemia cell infiltration in the livers, meninges, and kidneys of Ubc9 fl/+;Vav-cre mice compared to Ubc9 fl/+ mice. Transwell experiments indicated that Ubc9 deficiency resulted in increased migration of leukemia cells. Colony forming assay demonstrated that Ubc9-deficient leukemia cells formed significantly larger colonies. Flow cytometry analysis showed that Ubc9-deficient leukemia cells expressed high levels of c-kit and low levels of Mac1. To further elucidate the role of UBC9 in human AML cells, we constructed UBC9 deficient KG1 and K562 cell lines by using CRISPR-Cas9 technology. The UBC9 KD cell lines presented the same phenotype such as the increase of cell viability and the enhancement of invasiveness. To better understand the role that UBC9 plays in AML cell biology, we performed a system-level approach based on quantitative SUMO proteomic analysis to identify potential UBC9 substrates and then identified PARP1 with SUMOylation site lysine residue 486 (K486) by multiomics analysis. Finally, we validated that UBC9-mediated PARP1 SUMOylation played an important role in AML progression by activating the NF-kappa B pathway via the site-directed mutation of PARP1 (K486R) in UBC9-KO AML cells by using western blot and Co-immunoprecipitation. Together, our study revealed that UBC9 KO accelerated AML progression and enabled us to contrast global SUMOylation profiles of UBC9 in AML cells. In addition, we found that the PARP1 SUMOylation-mediated NF-κB pathway activation is a positive regulatory mechanism in the tumorigenesis of AML. These findings may provide potential clinical implications for AML treatment.