Protein Annexin Research Articles

A Nonfouling Electrochemical Biosensor for Protein Analysis in Complex Body Fluids Based on Multifunctional Peptide Conjugated with PEGlyated Phospholipid.

Developing antifouling biosensors capable of performing robustly in complex human body fluids is crucial for biomarker diagnosis and health monitoring. Herein, an antifouling and highly sensitive and stable biosensor was constructed through the self-assembly of the designed conjugates composed of a multifunctional peptide (MP) and PEGylated distearoylphosphatidylethanolamine (DSPE-PEG). The self-assembly capability of the DSPE-PEG-MP was demonstrated clearly through coarse-grained molecular dynamics simulation and transmission electron microscopy, and it can be effectively self-assembled onto the electrode surface modified with gold nanoparticles. The MP was designed to be antifouling and contained a peptide sequence that can specifically bind the target protein Annexin A1 (ANXA1), and the D-type amino acid composition of MP can enhance its resistance to enzymatic hydrolysis. The unique design of MP, in conjugation with the self-assembly capability of the PEGylated phospholipid DSPE-PEG, enabled the biosensor to exhibit excellent antifouling capability and stability in various complex human body fluids. The biosensor was capable of sensitively and selectively quantifying ANXA1 and achieved a limit of detection down to 0.12 pg mL-1. More importantly, the biosensor demonstrated satisfactory accuracy for ANXA1 detection in clinical serum samples, as verified by the enzyme linked immunosorbent assay (ELISA) kits. It is expected that various antifouling biosensors suitable for application in complex biological environments can be constructed by utilizing the strategy of designing similar DSPE-PEG-MP conjugates.

Soybean Agglutinin Induced Apoptotic Effects by Down-Regulating ANXA2 Through FAK Pathway in IPEC-J2 Cells.

Soybean agglutinin (SBA) is an anti-nutritional factor in soybean, possesses toxic effects by binding to intestinal epithelial cells, and finally interferes the digestion and absorption of nutrients in humans and animals. Annexin A2 (ANXA2) is one of the SBA-specific binding proteins in intestinal epithelial cells and participates in multiple cellular biological processes. However, whether SBA affects apoptosis through ANXA2 and its apoptosis-related pathway remains unclear. IPEC-J2 is an ideal model to study human intestinal health. Therefore, this study aims to investigate the effects of ANXA2 on SBA-induced intestinal epithelial cell apoptosis and the related pathway mechanism using IPEC-J2 as a cell model. The results showed that SBA induced the apoptosis through FAK signal pathway and decreased the gene and protein expressions of ANXA2 in IPEC-J2. The expression of ANXA2 protein had a negative correlation with the apoptosis rates, and a positive correlation with the expression of FAK protein and FAK pathway downstream proteins. In conclusion, SBA induced apoptosis of IPEC-J2 cells by downregulating the expression of ANXA2, which activated the FAK pathway. These findings highlight the toxic mechanism of SBA, which will provide basis for studying the toxicity mechanisms of other food-derived anti-nutrients and provide a new perspective for human gastrointestinal health and related cancer treatment.

Annexin A2 promotes proliferative vitreoretinopathy in response to a macrophage inflammatory signal in mice

Proliferative vitreoretinopathy is a vision-threatening response to penetrating ocular injury, for which there is no satisfactory treatment. In this disorder, retinal pigment epithelial cells, abandon their attachment to Bruch’s membrane on the scleral side of the retina, transform into motile fibroblast-like cells, and migrate through the retinal wound to the vitreal surface of the retina, where they secrete membrane-forming proteins. Annexin A2 is a calcium-regulated protein that, in complex with S100A10, assembles plasmin-forming proteins at cell surfaces. Here, we show that, in proliferative vitreoretinopathy, recruitment of macrophages and directed migration of retinal pigment epithelial cells are annexin A2-dependent, and stimulated by macrophage inflammatory protein-1α/β. These factors induce translocation of annexin A2 to the cell surface, thus enabling retinal pigment epithelial cell migration following injury; our studies reveal further that treatment of mice with intraocular antibody to either annexin A2 or macrophage inflammatory protein dampens the development of proliferative vitreoretinopathy in mice.

Homology-based identification and structural analysis of Pangasius hypophthalmus Annexins and Serine proteases to search molecules for wound healing applications

Chronic wounds and burns are a worldwide healthcare problem that erodes patients’ well-being and healthcare systems. This silent and costly epidemic requires new, cost-efficient solutions to improve patients’ physical and economic welfare. Eschar-degrading vegetal and bacterial proteases have been utilized as a solution. However, these proteins are evolutionarily far from those present in human wound healing. Serine protease (SP) and annexin (ANX) proteins interact within the skin healing process. A homology-based identification pipeline can help in discovering selective human SP and ANX analogs in the epithelial tissue of the fast-healing species, Pangasius hypophthalmus. In the present work, we found 14 candidates for RT-PCR in P. hypophthalmus using homology inference. The genetically detected candidates were then structurally and sequentially analyzed to understand their possible relation to SPs and ANXs involved in human wound healing. A total of six TBLASTN/BLASTX candidates (four SPs and two ANXs) were detected in P. hypophthalmus skin. Structural analysis revealed that all SP candidates resembled human KLK4, KLK5, KLK6, and KLK8, whereas all ANX only resembled human ANXA4. Structure and sequence analysis revealed high conservation of ANX Ca2+ binding sites (GDXD) and SP catalytic triad (HDS) motifs. In addition, structural analysis revealed that SP substrate selectivity position 186 was the main difference between human KLK5 and P. hypophthalmus SPs. These findings may allow the proposal and testing of more selective formulations, broadening treatments beyond debridement.

Decoding Octopus Skin Mucus: Impact of Aquarium-Maintenance and Senescence on the Proteome Profile of the Common Octopus (Octopus vulgaris).

The common octopus (Octopus vulgaris) is an excellent candidate for aquaculture diversification, due to its biological traits and high market demand. To ensure a high-quality product while maintaining welfare in captive environments, it is crucial to develop non-invasive methods for testing health biomarkers. Proteins found in skin mucus offer a non-invasive approach to monitoring octopus welfare. This study compares the protein profiles in the skin mucus of wild, aquarium-maintained, and senescent specimens to identify welfare biomarkers. A tandem mass tag (TMT) coupled with an Orbitrap Eclipse Tribrid mass spectrometer was used to create a reference dataset from octopus skin mucus, identifying 1496 non-redundant protein groups. Although similar profiles were observed, differences in relative abundances led to the identification of potential biomarkers, including caspase-3-like, protocadherin 4, deleted in malignant brain tumors, thioredoxin, papilin, annexin, cofilin and mucin-4 proteins. Some of these proteins also revealed potential as bioactive peptides. This investigation provides the most extensive analysis of the skin mucus proteome in the common octopus and is the first to explore how aquarium maintenance and senescence alter the mucus proteome. This research highlights the potential of skin mucus protein/peptides as non-invasive monitoring biomarkers in cultured animals.

GPCRs overexpression and impaired fMLP-induced functions in neutrophils from chronic kidney disease patients.

G-protein coupled receptors (GPCRs) expressed on neutrophils regulate their mobilization from the bone marrow into the blood, their half-live in the circulation, and their pro- and anti-inflammatory activities during inflammation. Chronic kidney disease (CKD) is associated with systemic inflammatory responses, and neutrophilia is a hallmark of CKD onset and progression. Nonetheless, the role of neutrophils in CKD is currently unclear. Blood and renal tissue were collected from non-dialysis CKD (grade 3 - 5) patients to evaluate GPCR neutrophil expressions and functions in CKD development. CKD patients presented a higher blood neutrophil-to-lymphocyte ratio (NLR), which was inversely correlated with the glomerular filtration rate (eGFR). A higher frequency of neutrophils expressing the senescent GPCR receptor (CXCR4) and activation markers (CD18+CD11b+CD62L+) was detected in CKD patients. Moreover, CKD neutrophils expressed higher amounts of GPCR formyl peptide receptors (FPR) 1 and 2, known as neutrophil pro- and anti-inflammatory receptors, respectively. Cytoskeletal organization, migration, and production of reactive oxygen species (ROS) by CKD neutrophils were impaired in response to the FPR1 agonist (fMLP), despite the higher expression of FPR1. In addition, CKD neutrophils presented enhanced intracellular, but reduced membrane expression of the protein Annexin A1 (AnxA1), and an impaired ability to secrete it into the extracellular compartment. Secreted and phosphorylated AnxA1 is a recognized ligand of FPR2, pivotal in anti-inflammatory and efferocytosis effects. CKD renal tissue presented a low number of neutrophils, which were AnxA1+. Together, these data highlight that CKD neutrophils overexpress GPCRs, which may contribute to an unbalanced aging process in the circulation, migration into inflamed tissues, and efferocytosis.

Pleomorphic adenoma and carcinoma ex-pleomorphic adenoma tumorigenesis: A proteomic analysis.

To conduct a comprehensive proteomic analysis of normal salivary gland tissue, pleomorphic adenoma (PA), and carcinoma ex-pleomorphic adenoma (CXPA), and validate the proteomic findings using immunohistochemistry. Six normal salivary gland tissues, seven PA and seven CXPA samples underwent laser microdissection followed by liquid chromatography coupled to mass spectrometry. Protein identification and quantification were performed using MaxQuant software. Statistical analysis and functional enrichment were conducted using the Perseus platform and STRING tool, respectively. Immunohistochemistry was used for validation. Comparative proteomic analysis revealed 2680 proteins across the three tissue types, with 799 significantly altered between groups. Translocation protein SEC63 homolog, Annexin A6 and Biglycan were up-regulated in CXPA compared to PA. Decorin was markedly up-regulated in both PA and CXPA compared to normal salivary gland (log2 fold changes of 7.58 and 7.38, respectively). Validation confirmed elevated levels of Biglycan and Decorin in the extracellular matrix of CXPA compared to PA. Proteomic analysis identified differential protein expression patterns associated with malignant transformation of PA into CXPA. Findings indicate a crucial role for extracellular matrix proteins, specifically Biglycan and Decorin, in the tumorigenic progression of PA and CXPA.

Zoledronic acid relieves steroid-induced avascular necrosis of femoral head via inhibiting FOXD3 mediated ANXA2 transcriptional activation

BackgroundZoledronic acid (ZOL) is a type of bisphosphonate with good therapeutic effects on orthopaedic diseases. However, the pharmacological functions of ZOL on steroid-induced avascular necrosis of femoral head (SANFH) and the underlying mechanism remain unclear, which deserve further research. MethodsSANFH models both in vivo and in vitro were established by dexamethasone (Dex) stimulation. Osteoclastogenesis was examined by TRAP staining. Immunofluorescence was employed to examine autophagy marker (LC3) level. Cell apoptosis was analyzed by TUNEL staining. The interaction between Foxhead box D3 protein (FOXD3) and Annexin A2 (ANXA2) promoter was analyzed using ChIP and dual luciferase reporter gene assays. ResultsDex aggravated osteoclastogenesis and induced osteoclast differentiation and autophagy in vitro, which was abrogated by ZOL treatment. PI3K inhibitor LY294002 abolished the inhibitory effect of ZOL on Dex-induced osteoclast differentiation and autophagy. FOXD3 overexpression neutralized the downregulation effects of ZOL on Dex-induced osteoclasts by transcriptionally activating ANXA2. ANXA2 knockdown reversed the effect of FOXD3 overexpression on ZOL-mediated biological effects in Dex-treated osteoclasts. In addition, ZOL improved SANFH symptoms in rats. ConclusionZOL alleviated SANFH through regulating FOXD3 mediated ANXA2 transcriptional activity and then promoting PI3K/AKT/mTOR pathway, revealing that FOXD3 might be a target for ZOL in SANFH treatment.

Hypoxia-Preconditioned BMSC-Derived Exosomes Induce Mitophagy via the BNIP3-ANAX2 Axis to Alleviate Intervertebral Disc Degeneration.

Intervertebral disc degeneration (IVDD) is a chronic degenerative disease involving the aging and loss of proliferative capacity of nucleus pulposus cells (NPCs), processes heavily dependent on mitochondrial dynamics and autophagic flux. This study finds that the absence of BCL2/adenovirus E1B 19kDa interacting protein 3 (BNIP3) is associated with senescence-related NPC degeneration, disrupting mitochondrial quality control. Bone marrow mesenchymal stem cells (BMSCs) have multidirectional differentiation potential and produce extracellular vesicles containing cellular activators. Therefore, in this study, BMSCs are induced under hypoxic stimulation to deliver BNIP3-rich extracellular vesicles to NPCs, thereby alleviating aging-associated mitochondrial autophagic flux, promoting damaged mitochondrial clearance, and restoring mitochondrial quality control. Mechanistically, BNIP3 is shown to interact with the membrane-bound protein annexin A2 (ANXA2), enabling the liberation of the transcription factor EB (TFEB) from the ANXA2-TFEB complex, promoting TFEB nuclear translocation, and regulating autophagy and lysosomal gene activation. Furthermore, a rat model of IVDD is established and verified the in vivo efficacy of the exosomes in repairing disc injuries, delaying NPC aging, and promoting extracellular matrix (ECM) synthesis. In summary, hypoxia-induced BMSC exosomes deliver BNIP3-rich vesicles to alleviate disc degeneration by activating the mitochondrial BNIP3/ANXA2/TFEB axis, providing a new target for IVDD treatment.

Generation of B7-H3 isoform regulated by ANXA2/NSUN2/YBX1 axis in human glioma.

In recent years, in the development of emerging immunotherapy, B7-H3 is also termed as CD276 and has become a novel chimeric antigen receptor (CAR)-T target against glioma and other tumours, and aroused extensive attention. However, B7-H3 has three isoforms (2, 3 and 4Ig) with the controversial expression and elusive function in tumour especially glioma. The current study mainly focuses on the regulatory factors and related mechanisms of generation of different B7-H3 isoforms. First, we have determined that 2Ig is dominant in glioma with high malignancy, and 4Ig is widely expressed, whereas 3Ig shows negative expression in all glioma. Next, we have further found that RNA binding protein annexin A2 (ANXA2) is essential for B7-H3 isoform maintenance, but fail to determine the choice of 4Ig or 2Ig. RNA methyltransferase NOP2/Sun RNA methyltransferase 2 (NSUN2) and 5-methylcytosine reader Y-box binding protein 1 (YBX1) facilitate the production of 2Ig. Our findings have uncovered a series of factors (ANXA2/NSUN2/YBX1) that can determine the alternative generation of different isoforms of B7-H3 in glioma. Our result aims to help peers gain a clearer understanding of the expression and regulatory mechanisms of B7H3 in tumour patients, and to provide better strategies for designing B7H3 as a target in immunotherapy.

Calcium influx rapidly establishes distinct spatial recruitments of Annexins to cell wounds.

To survive daily damage, the formation of actomyosin ring at the wound edge is required to rapidly close cell wounds. Calcium influx is one of the start signals for these cell wound repair events. Here, we find that the rapid recruitment of all 3 Drosophila calcium-responding and phospholipid-binding Annexin proteins (AnxB9, AnxB10, and AnxB11) to distinct regions around the wound is regulated by the quantity of calcium influx rather than their binding to specific phospholipids. The distinct recruitment patterns of these Annexins regulate the subsequent recruitment of RhoGEF2 and RhoGEF3 through actin stabilization to form a robust actomyosin ring. Surprisingly, while the wound does not close in the absence of calcium influx, we find that reduced calcium influx can still initiate repair processes, albeit leading to severe repair phenotypes. Thus, our results suggest that, in addition to initiating repair events, the quantity of calcium influx is important for precise Annexin spatiotemporal protein recruitment to cell wounds and efficient wound repair.

Novel protein drug conjugate ANXV-chemo and cytotoxic and anti-proliferative effects against human triple-negative breast cancer in vitro.

e15024 Background: Targeted delivery of cytotoxic drugs to tumor cells and tumor microenvironment remains a fundamental challenge in the fight against cancer including triple-negative breast cancer (TNBC). The nonapoptotic externalization of the phospholipid phosphatidylserine (PS) on cancer cells is an attractive biomarker. Cancer cells have up to 90-fold more PS molecules externalized than healthy cells, and TNBC cells have high PS externalization. The Annexin A5 human protein has a high affinity to PS and has shown anti-cancer effects both in vitro and in vivo. Here we utilize the recombinant protein Annexin A5 (ANXV), currently in Phase 2a as a treatment of retinal vein occlusion, as a delivery vehicle to deliver a covalently linked chemotherapy (chemo) to TNBC cells via externalized PS. Methods: Chemo was conjugated to ANXV through a one-step SMCC crosslinking reaction. ANXV-chemo was analyzed via LC-MS and the weighted average of the drug-to-ANXV ratio (DAR) was determined. MDA-MB-231 TNBC cells were utilized for internalization, cytotoxic, and proliferation studies. Incucyte was used to monitor live cells for up to 96 hours (samples tested in quadruplicate). For internalization studies, ANXV-pHrodo red was incubated with the cells, and the fluorescent intensity was measured over 48 hours. For the cytotoxic and proliferation studies, ANXV-chemo (0-100 nM), sacituzumab govitecan-hziy (SG) (0-100 nM), or thefree chemo (0-200nM) were incubated with the cells for up to 96 hours. All concentrations refer to the amount of drug linked to the conjugates or free drug. All data analysis was completed in GraphPad Prism (10.1.1). Results: ANXV-chemo was successfully crosslinked and had a DAR of 2.4 ± 0.23 chemo molecules. ANXV was quickly internalized, and ANXV could be detected within the cells for 48 hours after initial incubation, indicating constant cell uptake of the protein. The ANXV-chemo was nearly 30 times more cytotoxic to the TNBC cells than the free chemo as indicated by the EC50 after 72 hours (ANXV-chemo = 1.2 nM; free chemo = 36.7 nM). When comparing equal molar drug concentrations (4 nM), ANXV-chemo had more significant anti-proliferative and cytotoxic effects than the standard of care SG. By 24 hours, ANXV-chemo significantly increased cell death (p < 0.0021). By 48 hours, ANXV-chemo significantly decreased proliferation (p < 0.0332). After 96 hours, ANXV-chemo inhibited proliferation by 5.5 times (p < 0.0001) and induced 6.5 times more cell death (p < 0.0002) when compared to SG. Conclusions: By covalently linking chemo to ANXV, an efficient and PS-targeted treatment for TNBC has been developed. ANXV-chemo demonstrated significantly better anticancer effects in vitro when compared to the free drug and current targeted standard of care treatments. An investigation of ANXV-chemo as a targeted anticancer agent is currently underway in a murine TNBC model.

Shape of the membrane neck around a hole during plasma membrane repair

Plasma membrane damage and rupture occurs frequently in cells, and holes must be sealed rapidly to ensure homeostasis and cell survival. The membrane repair machinery is known to involve recruitment of curvature-inducing annexin proteins, but the connection between membrane remodeling and hole closure is poorly described. The induction of curvature by repair proteins leads to the possible formation of a membrane neck around the hole as a key intermediate structure before sealing. We formulate a theoretical model of equilibrium neck shapes to examine the potential connection to a repair mechanism. Using variational calculus, the shape equations for the membrane near a hole are formulated and solved numerically. The system is described under a condition of fixed area, and a shooting approach is applied to fulfill the boundary conditions at the free membrane edge. A state diagram of neck shapes is produced describing the variation in neck morphology with respect to the membrane area. Two distinct types of necks are predicted, one with conformations curved beyond π existing at positive excess area, whereas flat neck conformations (curved below π) have negative excess area. The results indicate that in cells, the supply of additional membrane area and a change in edge tension is linked to the formation of narrow and curved necks. Such necks may be susceptible to passive or actively induced membrane fission as a possible mechanism for hole sealing during membrane repair in cells.

Annexin-A5 monomer as a membrane repair agent for the treatment of renal ischemia-reperfusion injury.

Renal ischemia-reperfusion injury (IRI) is one of the causes of acute kidney injury. Annexin A5 (AnxA5), a calcium-dependent cell membrane-binding protein, shows protective effects in various organ IRI models. This study explored the therapeutic effect of exogenous AnxA5 monomer protein on renal IRI and its potential mechanism of action. Different doses of AnxA5 were injected intravenously to treat bilateral renal IRI in SD rats. This model confirmed the protective effects of AnxA5 on kidney structure and function. In vitro, HK-2 cells were subjected to hypoxia for 12h, followed by restoration of normal oxygen supply to simulate IRI. In vitro experiments demonstrated the mechanism of action of AnxA5 by measuring cellular activity and permeability. A comparison of the mutant AnxA5 protein M23 and the application of a calcium-free culture medium further validated the protective effect of AnxA5 by forming a network structure. Exogenous AnxA5 monomers prevented renal IRI by binding to the damaged renal tubular epithelial cell membrane, forming a two-dimensional network structure to maintain cell membrane integrity, and ultimately prevent cell death.

Characterization of antigenic proteins of the Taenia solium postoncospheral form

Neurocysticercosis is the leading cause for acquired epilepsy worldwide, and it is caused by the larval stage of the parasite Taenia solium. Several proteins of this stage have been characterized and studied to understand the parasite-host interaction, however, the proteins from the early cysticercus stages (the postoncospheral form) have not yet been characterized. The study of the postoncospheral form proteins is important to understand the host-parasite relationship in the early stages of infection. The aim of this work was to identify postoncospheral form antigenic proteins using sera from neurocysticercosis patients. T. solium activated oncospheres were cultured in HCT-8 cells to obtain the postoncospheral form. Soluble total and excretory/secretory proteins were obtained from the postoncospheral form and were incubated with both pool sera and individual serum of neurocysticercosis positive human patients. Immunoblotting showed target antigenic proteins with apparent molecular weights of 23 kDa and 46–48 kDa. The 46–48 kDa antigen bands present in soluble total and excretory/secretory postoncospheral form proteins were analyzed by LC-MS/MS; proteins identified were: nuclear elongation factor 1 alpha, enolase, unnamed protein product/antigen diagnostic GP50, calcium binding protein calreticulin precursor and annexin. The postoncospheral form expresses proteins related to interaction with the host, some of these proteins are predicted to be exosomal proteins. In conclusion, postoncospheral proteins are consistent targets of the humoral immune response in human and may serve as targets for diagnosis and vaccines.

METTL3-mediated deficiency of lncRNA HAR1A drives non-small cell lung cancer growth and metastasis by promoting ANXA2 stabilization

We previously reported lncRNA HAR1A as a tumor suppressor in non-small cell lung cancer (NSCLC). However, the delicate working mechanisms of this lncRNA remain obscure. Herein, we demonstrated that the ectopic expression of HAR1A inhibited the proliferation, epithelial-mesenchymal transition (EMT), migration, and invasion of NSCLC cells and enhanced paclitaxel (PTX) sensitivity in vitro and in vivo. We identified the oncogenic protein annexin 2 (ANXA2) as a potential interacting patterner of HAR1A. HAR1A overexpression enhanced ANXA2 ubiquitination and accelerated its degradation via the ubiquitin–proteasome pathway. We further uncovered that HAR1A promoted the interaction between E3 ubiquitin ligase TRIM65 and ANXA2. Moreover, the ANXA2 plasmid transfection could reverse HAR1A overexpression-induced decreases in proliferation, migration, and invasion of NSCLC cells and the activity of the NF-κB signaling pathway. Finally, we found that HAR1A loss in NSCLC might be attributed to the upregulated METTL3. The m6A modification levels of HAR1A were increased in cancer cells, while YTHDF2 was responsible for recognizing m6A modification in the HAR1A, leading to the disintegration of this lncRNA. In conclusion, we found that METTL3-mediated m6A modification decreased HAR1A in NSCLC. HAR1A deficiency, in turn, stimulated tumor growth and metastasis by activating the ANXA2/p65 axis.

The time-dependent expression of FPR2 and ANXA1 in murine deep vein thrombosis model and its relation to thrombus age.

Thrombus age determination in fatal venous thromboembolism cases is an important task for forensic pathologists. In this study, we investigated the time-dependent expressions of formyl peptide receptor 2 (FPR2) and Annexin A1 (ANXA1) in a stasis-induced deep vein thrombosis (DVT) murine model, with the aim of obtaining useful information for thrombus age timing. A total of 75 ICR mice were randomly classified into thrombosis group and control group. In thrombosis group, a DVT model was established by ligating the inferior vena cava (IVC) of mice, and thrombosed IVCs were harvested at 1, 3, 5, 7, 10, 14, and 21days after modeling. In control group, IVCs without thrombosis were taken as control samples. The expressions of FPR2 and ANXA1 during thrombosis were detected using immunohistochemistry and double immunofluorescence staining. Their protein and mRNA levels in the samples were determined by Western blotting and quantitative real-time PCR. The results reveal that FPR2 was predominantly expressed by intrathrombotic neutrophils and macrophages. ANXA1 expression in the thrombi was mainly distributed in neutrophils, endothelial cells of neovessels, and fibroblastic cells. After thrombosis, the expressions of FPR2 and ANXA1 were time-dependently up-regulated. The percentage of FPR2-positive cells and the level of FPR2 protein significantly elevated at 1, 3, 5 and 7 days after IVC ligation as compared to those at 10, 14 and 21 days after ligation (p < 0.05). Moreover, the mRNA level of FPR2 were significantly higher at 5 days than that at the other post-ligation intervals (p < 0.05). Besides, the levels of ANXA1 mRNA and protein peaked at 10 and 14 days after ligation, respectively. A significant increase in the mRNA level of ANXA1 was found at 10 and 14 days as compared with that at the other post-ligation intervals (p < 0.01). Our findings suggest that FPR2 and ANXA1 are promising as useful markers for age estimation of venous thrombi.

SUMOylation of annexin A6 retards cell migration and tumor growth by suppressing RHOU/AKT1–involved EMT in hepatocellular carcinoma

BackgroundThe protein annexin A6 (AnxA6) is involved in numerous membrane-related biological processes including cell migration and invasion by interacting with other proteins. The dysfunction of AnxA6, including protein expression abundance change and imbalance of post-translational modification, is tightly related to multiple cancers. Herein we focus on the biological function of AnxA6 SUMOylation in hepatocellular carcinoma (HCC) progression.MethodsThe modification sites of AnxA6 SUMOylation were identified by LC-MS/MS and amino acid site mutation. AnxA6 expression was assessed by immunohistochemistry and immunofluorescence. HCC cells were induced into the epithelial-mesenchymal transition (EMT)-featured cells by 100 ng/mL 12-O-tetradecanoylphorbol-13-acetate exposure. The ability of cell migration was evaluated under AnxA6 overexpression by transwell assay. The SUMO1 modified AnxA6 proteins were enriched from total cellular proteins by immunoprecipitation with anti-SUMO1 antibody, then the SUMOylated AnxA6 was detected by Western blot using anti-AnxA6 antibody. The nude mouse xenograft and orthotopic hepatoma models were established to determine HCC growth and tumorigenicity in vivo. The HCC patient’s overall survival versus AnxA6 expression level was evaluated by the Kaplan–Meier method.ResultsLys579 is a major SUMO1 modification site of AnxA6 in HCC cells, and SUMOylation protects AnxA6 from degradation via the ubiquitin-proteasome pathway. Compared to the wild-type AnxA6, its SUMO site mutant AnxA6K579R leads to disassociation of the binding of AnxA6 with RHOU, subsequently RHOU-mediated p-AKT1ser473 is upregulated to facilitate cell migration and EMT progression in HCC. Moreover, the SENP1 deSUMOylates AnxA6, and AnxA6 expression is negatively correlated with SENP1 protein expression level in HCC tissues, and a high gene expression ratio of ANXA6/SENP1 indicates a poor overall survival of patients.ConclusionsAnxA6 deSUMOylation contributes to HCC progression and EMT phenotype, and the combination of AnxA6 and SENP1 is a better tumor biomarker for diagnosis of HCC grade malignancy and prognosis.

Differential Expression of Annexin A1 Protein in Colorectal, Lung, and Liver Cancer Tissues

Differential Expression of Annexin A1 Protein in Colorectal, Lung, and Liver Cancer Tissues

Dry environment on the expression of lacrimal gland S100A9, Anxa1, and Clu in rats via proteomics.

To investigate the underlying mechanism of dry environment (autumn dryness) affecting the lacrimal glands in rats. Twenty Sprague-Dawley rats were randomly divided into two groups. The rats were fed in specific pathogen free environment as the control group (n=10), and the rats fed in dry environment as the dryness group (n=10). After 24d, lacrimal glands were collected from the rats. The tissues morphology was observed by hematoxylin-eosin (HE) staining. Tandem mass tags (TMT) quantitative proteomics analysis technology was used to screen the differential expressed proteins of lacrimal glands between the two groups, then bioinformatics analysis was performed. Further, the immunohistochemical (IHC) method was used to verify the target proteins. In dryness group, the lacrimal glands lobule atrophied, the glandular cavities enlarged, the sparse nuclear distribution and scattered inflammatory infiltration between the acinus were observed. The proteomics exhibited that a total of 195 up-regulated and 236 down-regulated differential expressed proteins screened from the lacrimal glands of rats. It was indicated that the biological processes (BP) of differential expressed proteins mainly included cell processes and single BP. The cellular compositions of differential expressed proteins mainly located in cells, organelles. The molecular functions of differential expressed proteins mainly included binding, catalytic activity. Moreover, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that the differential expressed proteins mainly involved lysosome, complement and coagulation cascade, and ribosome pathway. The IHC result verified that the up-regulated expression proteins of Protein S100A9 (S100A9), Annexin A1 (Anxa1), and Clusterin (Clu) in lacrimal glands of rats in dryness group were higher than control group. The up-regulated expression proteins of S100A9, Anxa1, and Clu may be the potential mechanisms of dry eye symptoms caused by dry environment. This study provides clues of dry environments causing eye-related diseases for further studies.