Phospholipid-binding Proteins Research Articles

Papillary thyroid cancer and its gene polymorphism; A molecular mechanistic perspective

Thyroid cancer stands as the predominant malignancy within the endocrine system, comprising about 1% of newly identified cancer instances. Papillary Thyroid Cancer (PTC) is the predominant form of thyroid cancer, representing 80% or more of thyroid malignancies. Thyroid carcinoma harbours assorted genetic alterations which are highly prevalent, several of these characteristics are unique to this form of cancer. The conventional oncogenic genetic modifications frequently observed in thyroid carcinoma encompass RAS mutations RET/PTC rearrangements and PAX8-peroxisome proliferator-activated receptor g (PPARg) fusion oncogene. The lately discovered activating mutation in BRAF (the gene for the B-type RAF kinase, BRAF) the most widespread genetic modification in thyroid cancer (30-83%). RKIP (RAF kinase inhibitory protein) had formerly been delineated as a phospholipid binding protein. Mammalian RKIP/PEBP differs from other identified proteins and its role is still being clarified. RKIP over-expression can inhibit MEK interaction with RAF-1 and B-RAF. It plays a role in thyroid cancer progression and lymph node metastasis. So, elucidating mutational profile and protein expression of above cell signalling molecules will be very useful in determining a proper therapeutic target for anti-cancer molecules. Given that tumors often possess numerous genetic and cell signalling abnormalities, thus inhibiting a single signalling pathway is often therapeutically inefficacious, more success could be foreseen with agents directed against multiple cellular pathways.By determining the genetic profile and protein expression of mentioned MAP Kinase pathway molecules new targets can be identified for chemotherapic drugs and novel strategies will be charted out to make modifications in the map kinase pathway with the aim to stop the occurrence and distant metastasis of thyroid cancer.

The structural properties of full-length annexin A11.

Annexin A11 (ANXA11) is a calcium-dependent phospholipid-binding protein belonging to the annexin protein family and implicated in the neurodegenerative amyotrophic lateral sclerosis. Structurally, ANXA11 contains a conserved calcium-binding C-terminal domain common to all annexins and a putative intrinsically unfolded N-terminus specific for ANXA11. Little is known about the structure and functions of this region of the protein. By analogy with annexin A1, it was suggested that residues 38 to 59 within the ANXA11 N-terminus could form a helical region that would be involved in interactions. Interestingly, this region contains residues that, when mutated, may lead to clinical manifestations. In the present study, we have studied the structural features of the full-length protein with special attention to the N-terminal region using a combination of biophysical techniques which include nuclear magnetic resonance and small angle X-ray scattering. We show that the N-terminus is intrinsically disordered and that the overall features of the protein are not markedly affected by the presence of calcium. We also analyzed the 38-59 helix hypothesis using synthetic peptides spanning both the wild-type sequence and clinically relevant mutations. We show that the peptides have a remarkable character typical of a native helix and that mutations do not alter the behaviour suggesting that they are required for interactions rather than being structurally important. Our work paves the way to a more thorough understanding of the ANXA11 functions.

The role of the annexin A protein family at the maternal-fetal interface.

Successful pregnancy requires the tolerance of the maternal immune system for the semi-allogeneic embryo, as well as a synchrony between the receptive endometrium and the competent embryo. The annexin family belongs to calcium-regulated phospholipid-binding protein, which functions as a membrane skeleton to stabilize the lipid bilayer and participate in various biological processes in humans. There is an abundance of the annexin family at the maternal-fetal interface, and it exerts a crucial role in embryo implantation and the subsequent development of the placenta. Altered expression of the annexin family and dysfunction of annexin proteins or polymorphisms of the ANXA gene are involved in a range of pregnancy complications. In this review, we summarize the current knowledge of the annexin A protein family at the maternal-fetal interface and its association with female reproductive disorders, suggesting the use of ANXA as the potential therapeutic target in the clinical diagnosis and treatment of pregnancy complications.

Generation and pathogenicity of autoantibodies associated to thrombosis and hemostasis

Many acquired bleeding and thrombotic complications are provoked by autoantibodies to blood coagulation factors, or to hemostasis inhibitors and regulatory proteins. If occurrence of those antibodies remains rare or ultra-rare, affected patients are not always well-identified and associated pathologies are not always understood. Today, autoantigens tend to be better characterized. New available methods allow investigating structural changes of body components, responsible for auto-immunization. This renders it possible to develop laboratory assays for detecting autoantibodies and estimating their blood concentration. This review analyzes the major autoantibodies reported to be associated with hemorrhagic or thrombotic pathologies and their possible inducing causes when known. Pathogenicity is strongly patient- and context-dependent and is related to autoantibodies’ concentration, avidity, and capacity to bind to autoantigen structures in-vivo, misdirecting the immune system to the own body’s cells or organs. Identification of autoantigens allows for developing laboratory methods for testing autoantibodies and following their evolution kinetics. In-vitro investigations concern functional assays, to evaluate autoantibody’s capacity to inhibit physiological activities, or autoantigen-capture-based assays to detect autoantibodies, like with enzyme-linked immuno-sorbent assay (ELISA) methods. Exploring patients with autoimmune complications remains difficult as few specific assays are available. They mainly concern diseases with the highest incidence, like anti-phospholipid antibodies, lupus anticoagulants, or heparin-dependent antibodies. The present understanding suggests that antibodies to ubiquitous components, like phospholipids or polysaccharides, are actually targeted to proteins with a strong affinity binding to those components: Autoantibodies are not directed to phospholipids, but to phospholipid-binding proteins, and heparin-dependent antibodies are not directed to anticoagulant polysaccharides, but to platelet factor 4. Most pathogenic autoantibodies are of immunoglobulin G (IgG) isotype, but in some cases, IgM or IgA isotypes can be involved. Identification and characterization of autoantibodies associated to hemorrhagic or thrombotic pathologies remains complex at the laboratory level, although they are of high relevance for the right management of concerned patients.

Comprehensive analysis of annexin gene family and its expression in response to branching architecture and salt stress in crape myrtle

BackgroundAnnexin (ANN) is calcium (Ca2+)-dependent and phospholipid binding protein family, which is involved in plant growth and development and response to various stresses. However, little known about ANN genes were identified from crape myrtle, an ornamental horticultural plant widely cultivated in the world.ResultsHere, 9 LiANN genes were identified from Lagerstroemia indica, and their characterizations and functions were investigated in L. indica for the first time. The LiANN genes were divided into 2 subfamilies. The gene structure, chromosomal location, and collinearity relationship were also explored. In addition, the GO annotation analysis of these LiANNs indicated that they are enriched in molecular functions, cellular components, and biological processes. Moreover, transcription factors (TFs) prediction analysis revealed that bHLH, MYB, NAC, and other TFs can interact with the LiANN promoters. Interestingly, the LiANN2/4/6–9 were demonstrated to play critical roles in the branching architecture of crape myrtle. Furthermore, the LiANN2/6/8/9 were differentially expressed under salt treatment, and a series of TFs regulating LiANN2/6/8/9 expression were predicted to play essential roles in salt resistance.ConclusionsThese results shed light on profile and function of the LiANN gene family, and lay a foundation for further studies of the LiANN genes.

Annexin A7 mediates lysosome repair independently of ESCRT-III.

Lysosomes are crucial organelles essential for various cellular processes, and any damage to them can severely compromise cell viability. This study uncovers a previously unrecognized function of the calcium- and phospholipid-binding protein Annexin A7 in lysosome repair, which operates independently of the Endosomal Sorting Complex Required for Transport (ESCRT) machinery. Our research reveals that Annexin A7 plays a role in repairing damaged lysosomes, different from its role in repairing the plasma membrane, where it facilitates repair through the recruitment of ESCRT-III components. Notably, our findings strongly suggest that Annexin A7, like the ESCRT machinery, is dispensable for membrane contact site formation within the newly discovered phosphoinositide-initiated membrane tethering and lipid transport (PITT) pathway. Instead, we speculate that Annexin A7 is recruited to damaged lysosomes and promotes repair through its membrane curvature and cross-linking capabilities. Our findings provide new insights into the diverse mechanisms underlying lysosomal membrane repair and highlight the multifunctional role of Annexin A7 in membrane repair.

Toxoplasma membrane inositol phospholipid binding protein TgREMIND is essential for secretory organelle function and host infection

Apicomplexan parasites possess specialized secretory organelles called rhoptries, micronemes, and dense granules that play a vital role in host infection. In this study, we demonstrate that TgREMIND, a protein found in Toxoplasma gondii, is necessary for the biogenesis of rhoptries and dense granules. TgREMIND contains a Fes-CIP4 homology-Bin/Amphiphysin/Rvs (F-BAR) domain, which binds to membrane phospholipids, as well as a novel uncharacterized domain that we have named REMIND (regulator of membrane-interacting domain). Both the F-BAR domain and the REMIND are crucial for TgREMIND functions. When TgREMIND is depleted, there is a significant decrease in the abundance of dense granules and abnormal transparency of rhoptries, leading to a reduction in protein secretion from these organelles. The absence of TgREMIND inhibits host invasion and parasite dissemination, demonstrating that TgREMIND is essential for the proper function of critical secretory organelles required for successful infection by Toxoplasma.

MARCKS and PI(4,5)P2 reciprocally regulate actin-based dendritic spine morphology.

Myristoylated, alanine-rich C-kinase substrate (MARCKS) is an F-actin and phospholipid binding protein implicated in numerous cellular activities, including the regulation of morphology in neuronal dendrites and dendritic spines. MARCKS contains a lysine-rich effector domain that mediates its binding to plasma membrane phosphatidylinositol-4,5-biphosphate (PI(4,5)P2) in a manner controlled by PKC and calcium/calmodulin. In neurons, manipulations of MARCKS concentration and membrane targeting strongly affect the numbers, shapes, and F-actin properties of dendritic spines, but the mechanisms remain unclear. Here, we tested the hypothesis that the effects of MARCKS on dendritic spine morphology are due to its capacity to regulate the availability of plasma membrane PI(4,5)P2. We observed that the concentration of free PI(4,5)P2 on the dendritic plasma membrane was inversely proportional to the concentration of MARCKS. Endogenous PI(4,5)P2 levels were increased or decreased, respectively, by acutely overexpressing either phosphatidylinositol-4-phosphate 5-kinase (PIP5K) or inositol polyphosphate 5-phosphatase (5ptase). PIP5K, like MARCKS depletion, induced severe spine shrinkage; 5ptase, like constitutively membrane-bound MARCKS, induced aberrant spine elongation. These phenotypes involved changes in actin properties driven by the F-actin severing protein cofilin. Collectively, these findings support a model in which neuronal activity regulates actin-dependent spine morphology through antagonistic interactions of MARCKS and PI(4,5)P2.

Discovery of Unexpected Phospholipid-Binding Proteins in Antiphospholipid Syndrome Patients

INTRODUCTION: The antiphospholipid syndrome (APS), initially described nearly 4 decades ago, remains enigmatic with unclear pathophysiology. In addition to the lack of a mechanistic diagnostic test, APS exhibits considerable phenotypic heterogeneity, including the occurrence of “non-consensus” clinical manifestations (i.e. other than vascular thrombosis and pregnancy complications). Despite the evidence that phospholipid-binding factors are highly likely to be central to APS pathogenesis, the full complement of phospholipid-binding proteins has yet to be systemically assayed. Proteomic profiling by label-free quantitation (LFQ) may identify patterns of protein binding to phospholipid that are central to APS pathogenesis and thereby elucidate a basis for the heterogeneous clinical presentations of APS. METHODS: C18 silica beads were coated with phospholipid by incubation with a suspension of 30% phosphatidylserine/70% phosphatidylcholine. Plasmas from 4 groups: 1) APS patients (n=11), 2) APL positive patients who lacked clinical manifestations (“APL”) (n=11), 3) non-APL patients with thrombosis (“nonAPL”)(n=11), and 4) normal healthy controls (“normal”) (n=6) were incubated with the phospholipid-coated beads, and eluates were processed by gel electrophoresis followed by tryptic digestion. The resulting peptides were analyzed by LC-MS/MS on a Thermo Orbitrap Fusion Lumos. Protein identification, false discovery rate (FDR) estimation, and LFQ were performed using Proteome Discoverer 2.2 (Thermo). LFQ values were normalized by overall peptide abundance, and relative quantitation was obtained. Groupwise comparisons were performed using PD 2.2 in order to identify differentially enriched proteins. RESULTS: A total of 1,210 proteins were identified, with 875 proteins identified at high confidence (q<0.01). Based on gel electrophoresis, all samples had comparable amounts of total protein, and normalization of LFQ data on the basis of total proteins was therefore performed. Differentially expressed proteins between groups were identified using both fold-change and -log p value cutoffs (Figure 1a). Primary analysis focused on identifying proteins that were consistently preferentially bound from the APS plasmas compared to the normal controls. Novel findings included the following proteins related to coagulation, immune response, and to lipid binding that bound preferentially from APS plasmas compared to normal plasmas (APS:control ratios shown in parentheses): coagulation factor VII (10.4), heparin cofactor 2 (9.6), CD14 (6.1), apolipoprotein L1 (6.0), and apolipoprotein L2 (5.5). Additional proteins that preferentially bound from APS plasmas included OAF (13.7), BRSK1 (13.2), MINPP1 (12.5), fetuin B (10.5), SHBG (8.9), ADAMTS-like protein 4 (8.5), SERPIN A4 (7.6), alpha-2-HS glycoprotein (6.7) and inter-alpha-trypsin inhibitor heavy chains (H3, H4 and H1; 8.5, 6.6, 6.5 respectively). Consistent with prior knowledge of APS, and validating this LFQ approach immunoglobulins and complement proteins were also among the preferentially bound proteins and included: Ig heavy chain 1-3 (15.6), complement C9 (13.3), complement C4-B, (7.1), complement component C8 beta chain (5.6), Ig kappa variable 1D-8 (5.6), and Ig lambda-like polypeptide 1 (3.9). Interestingly, a number of identified proteins showed intermediate abundance (between APS and control) in the APL positive patients without clinical manifestations (“APL”) and APL negative patients with thrombosis (“nonAPL”) (see figure 1B-D for examples), suggesting a potential dose response effect. CONCLUSIONS: 1) These results are a first demonstration of concept for the ability of LFQ proteomics to address the complexity of APS through analysis of phospholipid-bound proteins. 2) A number of phospholipid-binding proteins, many of which have not been previously implicated in APS pathogenesis, are preferentially present in APS patient plasma 3) Some plasmas from APL and non-APL patients also show increased binding of some of these proteins compared to the normal controls, suggesting that LFQ may identify markers for subgroups of patients within these 2 categories.

Domain Specificity of Anti-Prothrombin Antibodies That Express Lupus Anticoagulant Activity

Background: The antiphospholipid syndrome (APS) is an autoimmune disorder characterized by the persistent presence of antiphospholipid antibodies (aPL). aPL are a heterogeneous group of autoantibodies directed against negatively charged phospholipids or phospholipid-binding proteins. Some of these autoantibodies exert lupus anticoagulant (LAC) activity, a laboratory phenomenon characterized by the prolongation of phospholipid-dependent coagulation tests. Recently, our group identified that LAC-positive anti-prothrombin antibodies activate platelets via the platelet receptor, FcγRIIA. However, a lot is still unclear regarding the mechanism by which these antibodies cause LAC and why some, but not all, antibodies in this heterogeneous group express LAC activity. Furthermore, experimental data on the binding site of these pathogenic anti-prothrombin antibodies are lacking. Aims: In this study we aimed to investigate the binding domain of LAC-positive monoclonal anti-prothrombin antibodies and APS patient derived anti-prothrombin antibodies. Method: Recombinant fragments of prothrombin were designed and produced in Chinese hamster ovary cells. Prothrombin fragment 1, fragment 2 and fragment 1.2 were coated on ELISA plates to study epitope specificity. Two LAC-positive monoclonal anti-prothrombin antibodies (3B1 and 28F4), two LAC-negative anti-prothrombin monoclonal antibodies (11H2 and 9C6), isolated anti-prothrombin antibodies derived from APS patients and plasma from APS patients were studied. A modified dilute Russell's viper venom time (DRVVT) test was used to evaluate whether the recombinant prothrombin constructs could neutralize the effect of LAC-positive monoclonal and patient derived anti-prothrombin antibodies. Furthermore, a fragment 1.2 nanobody was produced by immunizing Llama's followed by phage display. The ability of this nanobody to compete with LAC-positive monoclonal and patient derived anti-prothrombin antibodies for the prothrombin binding site was tested using a modified DRVVT test. Results: LAC-positive monoclonal anti-prothrombin antibodies recognized prothrombin fragment 2. Interestingly, LAC-negative anti-prothrombin antibodies bound exclusively to prothrombin fragment 1. In addition, LAC-ratios of patient samples correlated significantly with anti-fragment 2 antibody levels ( p<0.05, ρ=0.33), but not with anti-fragment 1 antibody levels. Furthermore, incubation with the prothrombin F1.2 nanobody almost completely reversed the prolonged DRVVT clotting time in plasma containing monoclonal and patient derived LAC-positive anti-prothrombin antibodies, but not in plasma containing LAC-positive anti-β2GP1 antibodies. This neutralising effect was also observed when the monoclonal anti-prothrombin antibody was incubated with prothrombin fragment 2. In contrast, the LAC-activity of anti-prothrombin antibodies was not inhibited when incubated with prothrombin fragment 1. Conclusion: Our data show that LAC-positive anti-prothrombin antibodies react with fragment 2 of prothrombin. Furthermore, a novel nanobody directed against prothrombin fragment 1.2 and prothrombin fragment 2 were able to neutralize a prothrombin-dependent LAC.

Cardiovascular risk in rare autoimmune diseases, lessons from a primary antiphospholipid syndrome patients

Abstract Background Antiphospholipid syndrome is a thrombo-inflammatory disease driven by circulating autoantibodies (aPL) that recognize cell surface phospholipids and phospholipid-binding proteins. In the absence of an underlying connective tissue disorder, the persistent presence of these antibodies associated with recurrent fetal losses and arterial or venous thrombosis is defined as a primary antiphospholipid syndrome (PAPS) with a prevalence of 1/2000 people, predominantly female. Purpose Estimation of cardiovascular risk presence based on markers of subclinical cardiovascular impairment. Methods 100 PAPS patients, with an average age of 47.70±13.14y, 93.7% females, were included in the study. Anticardiolipin antibodies (aCL IgG/IgM), anti-ß2 glycoprotein-I (anti-ß2GPI IgG/IgM), and lupus anticoagulant (LAC) were determined. Abnormal cut-off values used for left ventricular diastolic dysfunction (LVDD) were septal E ́<7 cm/sec, lateral E ́ <10 cm/sec, average E/E ́ ratio >14, LA volume index (LAVI) >34 mL/m2, and peak tricuspid regurgitation velocity >2.8 m/sec. LVDD was present if more than half parameters were with abnormal values. Endothelial dysfunction was assessed through ultrasound measurement of brachial artery flow-mediated dilation (FMD) and atherosclerosis progression by ultrasound assessment of intima-media thickness (cIMT) on carotid arteries (common carotid artery, CCA, internal carotid artery, ICA). The results were compared to 90 healthy, age and sex-matched controls. Results LVDD was significantly more prevalent in PAPS patients compared to healthy controls (24.8% vs. 2.2%, p=0.001). FMD was significantly lower in PAPS patients (10.76±5.15% vs. 14.20±6.76%, p=0.0001) and cIMT values were significantly higher (left ICA 0.80±0.29mm vs 0.56±0.20mm, p=0.001). All markers of subclinical atherosclerosis were significantly related to age and standard atherosclerotic risk factors present. Regarding aPL profile, anti-ß2GPI IgG positive PAPS patients had higher levels of cIMT ICA left (p=0.049) and LAC positive higher prevalence of LVDD (p=0.047) CONCLUSIONS: PAPS patients impose significantly higher prevalence of subclinical cardiovascular involvement compare to healthy population. Timely recognition, along with vigorous control of standard atherosclerotic risk factors and a proper therapeutic (antithrombotic and/or anticoagulant) regime, is of great importance.

Copine7 deficiency leads to hepatic fat accumulation via mitochondrial dysfunction

ObjectiveMitochondrial dysfunction affects hepatic lipid homeostasis and promotes ROS generation. Copine7 (CPNE7) belongs to the ubiquitous copine family of calcium-dependent phospholipid binding proteins. CPNE7 has a high calcium ion binding affinity and the capacity to scavenge reactive oxygen species (ROS). A recent study reported that abnormalities in fatty acid and lipid metabolism were linked to the gene variant of CPNE7. Therefore, the purpose of this study is to examine the role of Cpne7 in hepatic lipid metabolism based on mitochondrial function. MethodsLipid metabolism, mitochondrial function, and ROS production were investigated in high-fat diet (HFD)-fed Cpne7−/− mice and H2O2-damaged HepG2 hepatocytes following CPNE7 silencing or overexpression. ResultsCpne7 deficiency promoted severe hepatic steatosis in the HFD-induced NAFLD model. More importantly, mitochondrial dysfunction was observed along with an imbalance of mitochondrial dynamics in the livers of HFD-fed Cpne7−/−mice, resulting in high ROS levels. Similarly, CPNE7-silenced HepG2 hepatocytes showed high ROS levels, mitochondrial dysfunction, and increased lipid contents. On the contrary, CPNE7-overexpressed HepG2 cells showed low ROS levels, enhanced mitochondrial function and decreased lipid contents under H2O2-induced oxidative stress. ConclusionsIn the liver, Cpne7 deficiency causes excessive ROS formation and mitochondrial dysfunction, which aggravates lipid metabolism abnormalities. These findings provide evidence that Cpne7 deficiency contributes to the pathogenesis of NAFLD, suggesting Cpne7 as a novel therapeutic target for NAFLD.

Phase Separation and Fibrillization of Human Annexin A7 Are Mediated by Its Proline-Rich Domain.

Human annexin A7, a calcium- and phospholipid-binding protein, governs calcium homeostasis, plasma membrane repair, apoptosis, and tumor progression. A7 contains an N-terminal proline-rich domain (PRD; 180 residues, ∼24% prolines) that determines its functional specificity. Using microscopy and dye-binding assays, we show that recombinant A7 and its isolated PRD spontaneously phase separate into spherical condensates, which subsequently transform into β-sheet-rich fibrils. We demonstrate that fibrillization of A7-PRD proceeds via primary nucleation and fibril-catalyzed secondary nucleation processes, as determined by chemical kinetics, providing a mechanistic basis for its amyloid assembly. This study confirms and highlights a subclass of eukaryotic PRDs prone to forming aggregates with important physiological and pathological implications.

Methyl viologen-induced changes in the Arabidopsis proteome implicate PATELLIN 4 in oxidative stress responses.

The photosynthesis-induced accumulation of reactive oxygen species in chloroplasts can lead to oxidative stress, triggering changes in protein synthesis, degradation, and the assembly/disassembly of protein complexes. Using shot-gun proteomics, we identified methyl viologen-induced changes in protein abundance in wild-type Arabidopsis and oxidative stress-hypersensitive fsd1-1 and fsd1-2 knockout mutants, which are deficient in IRON SUPEROXIDE DISMUTASE 1 (FSD1). The levels of proteins that are localized in chloroplasts and the cytoplasm were modified in all lines treated with methyl viologen. Compared with the wild-type, fsd1 mutants showed significant changes in metabolic protein and chloroplast chaperone levels, together with increased ratio of cytoplasmic, peroxisomal, and mitochondrial proteins. Different responses in proteins involved in the disassembly of photosystem II-light harvesting chlorophyll a/b binding proteins were observed. Moreover, the abundance of PATELLIN 4, a phospholipid-binding protein enriched in stomatal lineage, was decreased in response to methyl viologen. Reverse genetic studies using patl4 knockout mutants and a PATELLIN 4 complemented line indicate that PATELLIN 4 affects plant responses to oxidative stress by effects on stomatal closure.

AnnexinA6: a potential therapeutic target gene for extracellular matrix mineralization.

The mineralization of the extracellular matrix (ECM) is an essential and crucial process for physiological bone formation and pathological calcification. The abnormal function of ECM mineralization contributes to the worldwide risk of developing mineralization-related diseases; for instance, vascular calcification is attributed to the hyperfunction of ECM mineralization, while osteoporosis is due to hypofunction. AnnexinA6 (AnxA6), a Ca2+-dependent phospholipid-binding protein, has been extensively reported as an essential target in mineralization-related diseases such as osteoporosis, osteoarthritis, atherosclerosis, osteosarcoma, and calcific aortic valve disease. To date, AnxA6, as the largest member of the Annexin family, has attracted much attention due to its significant contribution to matrix vesicles (MVs) production and release, MVs-ECM interaction, cytoplasmic Ca2+ influx, and maturation of hydroxyapatite, making it an essential target in ECM mineralization. In this review, we outlined the recent advancements in the role of AnxA6 in mineralization-related diseases and the potential mechanisms of AnxA6 under normal and mineralization-related pathological conditions. AnxA6 could promote ECM mineralization for bone regeneration in the manner described previously. Therefore, AnxA6 may be a potential osteogenic target for ECM mineralization.

Knockdown of ANXA10 induces ferroptosis by inhibiting autophagy-mediated TFRC degradation in colorectal cancer

Annexin A10 (ANXA10) belongs to a family of membrane-bound calcium-dependent phospholipid-binding proteins, but its precise function remains unclear. Further research is required to understand its role in sessile serrated lesions (SSL) and colorectal cancer (CRC). We conducted transcriptome sequencing on pairs of SSL and corresponding normal control (NC) samples. Bioinformatic methods were utilized to assess ANXA10 expression in CRC. We knocked down and overexpressed ANXA10 in CRC cells to examine its effects on cell malignant ability. The effect of ANXA10 on lung metastasis of xenograft tumor cells in nude mice was also assessed. Furthermore, we used quantitative polymerase chain reaction, western blotting, and flow cytometry for reactive oxygen species (ROS), lipid ROS, and intracellular Fe2+ to measure ferroptosis. Immunoblotting and Immunofluorescence staining were used to detect autophagy. We found that ANXA10 was significantly overexpressed in SSL compared to NC. ANXA10 was also highly expressed in BRAF mutant CRCs and was associated with poor prognosis. ANXA10 knockdown reduced the survival, proliferation, and migration ability of CRC cells. Knockdown of ANXA10 inhibited lung metastasis of CRC cells in mice. ANXA10 knockdown increased transferrin receptor (TFRC) protein levels and led to downregulation of GSH/GSSG, increased Fe2+, MDA concentration, and ROS and lipid ROS in cells. Knockdown of ANXA10 inhibited TFRC degradation and was accompanied by an accumulation of autophagic flux and an increase in SQSTM1. Finally, Fer-1 rescued the migration and viability of ANXA10 knockdown cell lines. In brief, the knockdown of ANXA10 induces cellular ferroptosis by inhibiting autophagy-mediated TFRC degradation, thereby inhibiting CRC progression. This study reveals the mechanism of ANXA10 in ferroptosis, suggesting that it may serve as a potential therapeutic target for CRC of the serrated pathway.

Oxidative Stress as a Regulatory Checkpoint in the Production of Antiphospholipid Autoantibodies: The Protective Role of NRF2 Pathway.

Oxidative stress is a well-known hallmark of Antiphospholipid Antibody Syndrome (APS), a systemic autoimmune disease characterized by arterial and venous thrombosis and/or pregnancy morbidity. Oxidative stress may affect various signaling pathways and biological processes, promoting dysfunctional immune responses and inflammation, inducing apoptosis, deregulating autophagy and impairing mitochondrial function. The chronic oxidative stress and the dysregulation of the immune system leads to the loss of tolerance, which drives autoantibody production and inflammation with the development of endothelial dysfunction. In particular, anti-phospholipid antibodies (aPL), which target phospholipids and/or phospholipid binding proteins, mainly β-glycoprotein I (β-GPI), play a functional role in the cell signal transduction pathway(s), thus contributing to oxidative stress and thrombotic events. An oxidation-antioxidant imbalance may be detected in the blood of patients with APS as a reflection of disease progression. This review focuses on functional evidence highlighting the role of oxidative stress in the initiation and progression of APS. The protective role of food supplements and Nuclear Factor Erythroid 2-Related Factor 2 (NRF2) activators in APS patients will be summarized to point out the potential of these therapeutic approaches to reduce APS-related clinical complications.

SYST-16 ANXA5 PROMOTES GLIOMA PROGRESSION THROUGH THE FAK/PI3K/AKT PATHWAY

Abstract Annexin A5 (ANXA5) is a member of the calcium dependent phospholipid binding protein family and participates in the regulation of a variety of physiological and pathological processes. However, whether and how ANXA5 affects the growth and progression of glioma remains unclear.We analyzed the expression of ANXA5 in multiple public databases, and verified the expression in 50 glioma clinical samples with Western blotting to study the correlation between ANXA5 and prognosis, survival, and clinical characteristics. In vitro experiments, we up-regulated and down-regulated the expression of ANXA5 in glioma cells, then treated glioma cells with up regulated ANXA5 expression with Y397 FAK specific phosphorylation inhibitor Y15, and verified the effect of ANXA5 on proliferation, migration and invasion of glioma cells through CCK8, clone formation, EDU, migration and invasion assays, scratching,and Western blotting proved that the expression level of ANXA5 affected the protein level of upstream and downstream factors of FAk/PI3K/AK pathway. ANXA5 is highly expressed in glioma and is related to clinical features such as grade, age and molecular typing. The higher the expression of ANXA5, the worse the prognosis. The increased expression of ANXA5 can promote the proliferation, migration and invasion of glioma cells, activate FAk/PI3K/AK pathway, and Y15 can reverse the promotion of ANXA5 overexpression; when ANXA5 is knocked down, the result is opposite. ANXA5 is highly expressed in glioma and is an independent prognostic factor of glioma patients. ANXA5 can promote the proliferation, migration and invasion of glioma cells through FAk/PI3K/AK pathway, and this promotion can be reversed by Y15.

Abstract P3050: Cpne5 Is Necessary For Normal Murine Sinoatrial Node Function In Vivo

Cpne5 ( Copine 5 ) is a Ca 2+ -dependent phospholipid binding protein implicated in membrane trafficking but has no known role within the heart to date. In single-cell RNA-sequencing of the developing mouse heart, we previously found Cpne5 gene expression to be highly enriched in the cardiac conduction system (CCS). Further, variants in CPNE5 were identified in 6 independent GWAS related to heart rate variability in humans. We therefore hypothesized that Cpne5 is necessary for normal CCS function through Ca 2+ -dependent membrane trafficking of ion channels. Whole mount immunostaining with 3D volumetric imaging (iDISCO+) of intact WT hearts demonstrated robust and specific protein expression of Cpne5 throughout all CCS components, including the sinoatrial node (SAN) and SAN-transitional cells. Next, to assess the necessity of Cpne5 within the CCS, systemic knockout mice (Cpne5KO) were generated using CRISPR/Cas9. Successful deletion of Cpne5 in CRISPR-generated KO mice was confirmed by PCR and, at the protein level, immunofluorescence. Homozygous Cpne5KO mice were viable, showed normal growth and allele frequencies segregated in a normal Mendelian fashion. No gross anatomic defects of the CCS were observed in Cpne5KO mice and transthoracic echocardiogram studies showed a slight reduction in left ventricular fractional shortening but otherwise a structurally normal heart. Notably, while surface electrocardiogram (ECG) demonstrated normal intervals (PR, QRS, QTc), Cpne5KO mice exhibited pronounced sinus arrhythmia, with notable sinus pauses, sinus bradycardia, and increased heart rate variability as compared to WT littermate controls on long term telemetry. Systemic loss-of-function of Cpne5 in mice resulted in marked sinus node dysfunction, consistent with human GWAS data, implicating a novel role for Cpne5 in CCS function and/or development. Additional phenotyping and molecular studies, including CPNE5 deletion in human induced-pluripotent cell-derived SAN-like cells, are ongoing to further elucidate its underlying mechanism.

Interaction of the antiphospholipid syndrome autoantigen beta-2 glycoprotein I with DNA and neutrophil extracellular traps

Beta-2 glycoprotein I (β2GPI) is a phospholipid-binding plasma protein and prominent autoantigen in antiphospholipid syndrome (APS). Here, we tested the hypothesis that β2GPI might bind to not only phospholipids, but also cell-free DNA and neutrophil extracellular traps (NETs). We report that β2GPI interacts with cell-free DNA from different species, as well as NETs, in a dose-dependent manner, retarding their migration in an agarose-gel electrophoretic mobility shift assay. We confirm the direct binding interaction of β2GPI with DNA and NETs by ELISA. We also demonstrate that β2GPI colocalizes with NET strands by immunofluorescence microscopy. Finally, we provide evidence that β2GPI-DNA complexes can be detected in the plasma of APS patients, where they positively correlate with an established biomarker of NET remnants. Taken together, our findings indicate that β2GPI interacts with DNA and NETs, and suggest that this interaction may play a role as a perpetuator and/or instigator of autoimmunity in APS.