SMARCA3, a chromatin-remodeling factor, is required for p11-dependent antidepressant action.

p11, through unknown mechanisms, is required for behavioral and cellular responses to selective serotonin reuptake inhibitors (SSRIs). We show that SMARCA3, a chromatin-remodeling factor, is a target for the p11/annexin A2 heterotetrameric complex. Determination of the crystal structure indicates that SMARCA3 peptide binds to a hydrophobic pocket in the heterotetramer. Formation of this complex increases the DNA-binding affinity of SMARCA3 and its localization to the nuclear matrix fraction. In the dentate gyrus, both p11 and SMARCA3 are highly enriched in hilar mossy cells and basket cells. The SSRI fluoxetine induces expression of p11 in both cell types and increases the amount of the ternary complex of p11/annexin A2/SMARCA3. SSRI-induced neurogenesis and behavioral responses are abolished by constitutive knockout of SMARCA3. Our studies indicate a central role for a chromatin-remodeling factor in the SSRI/p11 signaling pathway and suggest an approach to the development of improved antidepressant therapies. PAPERCLIP:

Multi-Omics Profiling Identifies Microglial Annexin A2 as a Key Mediator of NF-κB Pro-inflammatory Signaling in Ischemic Reperfusion Injury.

Cerebral stroke is one of the leading causes of mortality and disability worldwide. Restoring the cerebral circulation following a period of occlusion and subsequent tissue oxygenation leads to reperfusion injury. Cerebral ischemic reperfusion (I/R) injury triggers immune and inflammatory responses, apoptosis, neuronal damage, and even death. However, the cellular function and molecular mechanisms underlying cerebral I/R-induced neuronal injury are incompletely understood. By integrating proteomic, phosphoproteomic, and transcriptomic profiling in mouse hippocampi after cerebral I/R, we revealed that the differentially expressed genes and proteins mainly fall into several immune inflammatory response-related pathways. We identified that Annexin 2 (Anxa2) was exclusively upregulated in microglial cells in response to cerebral I/R in vivo and oxygen-glucose deprivation and reoxygenation (OGD/R) in vitro. RNA-seq analysis revealed a critical role of Anxa2 in the expression of inflammation-related genes in microglia via the NF-κB signaling. Mechanistically, microglial Anxa2 is required for nuclear translocation of the p65 subunit of NF-κB and its transcriptional activity upon OGD/R in BV2 microglial cells. Anxa2 knockdown inhibited the OGD/R-induced microglia activation and markedly reduced the expression of pro-inflammatory factors, including TNF-α, IL-1ß, and IL-6. Interestingly, conditional medium derived from Anxa2-depleted BV2 cell cultures with OGD/R treatment alleviated neuronal death in vitro. Altogether, our findings revealed that microglia Anxa2 plays a critical role in I/R injury by regulating NF-κB inflammatory responses in a non-cell-autonomous manner, which might be a potential target for the neuroprotection against cerebral I/R injury.

Accumulation of annexin A2 and S100A10 prevents apoptosis of apically delaminated, transformed epithelial cells.

In various epithelial tissues, the epithelial monolayer acts as a barrier. To fulfill its function, the structural integrity of the epithelium is tightly controlled. When normal epithelial cells detach from the basal substratum and delaminate into the apical lumen, the apically extruded cells undergo apoptosis, which is termed anoikis. In contrast, transformed cells often become resistant to anoikis and able to survive and grow in the apical luminal space, leading to the formation of multilayered structures, which can be observed at the early stage of carcinogenesis. However, the underlying molecular mechanisms still remain elusive. In this study, we first demonstrate that S100A10 and ANXA2 (Annexin A2) accumulate in apically extruded, transformed cells in both various cell culture systems and murine epithelial tissues in vivo. ANXA2 acts upstream of S100A10 accumulation. Knockdown of ANXA2 promotes apoptosis of apically extruded RasV12-transformed cells and suppresses the formation of multilayered epithelia. In addition, the intracellular reactive oxygen species (ROS) are elevated in apically extruded RasV12 cells. Treatment with ROS scavenger Trolox reduces the occurrence of apoptosis of apically extruded ANXA2-knockdown RasV12 cells and restores the formation of multilayered epithelia. Furthermore, ROS-mediated p38MAPK activation is observed in apically delaminated RasV12 cells, and ANXA2 knockdown further enhances the p38MAPK activity. Moreover, the p38MAPK inhibitor promotes the formation of multilayered epithelia of ANXA2-knockdown RasV12 cells. These results indicate that accumulated ANXA2 diminishes the ROS-mediated p38MAPK activation in apically extruded transformed cells, thereby blocking the induction of apoptosis. Hence, ANXA2 can be a potential therapeutic target to prevent multilayered, precancerous lesions.

LncNFYB promotes the proliferation of rheumatoid arthritis fibroblast-like synoviocytes via LncNFYB/ANXA2/ERK1/2 axis.

Long noncoding RNAs (lncRNAs) are specifically expressed in different diseases and regulate disease progression. To explore the functions of rheumatoid arthritis (RA)-specific lncRNA, we determined the lncRNA expression profile of fibroblast-like synoviocytes (FLS) obtained from patients with RA and osteoarthritis (OA) using a LncRNA microarray and identified up-regulated LncNFYB in RA as a potential therapeutic target. Using gain- and loss-of-function studies, LncNFYB was proven to promote FLS proliferation and cell cycle progress but not affect their invasion, migration, and apoptotic abilities. Further investigation discovered that LncRNA could combine with annexin A2 (ANXA2) and enhance the level of phospho-ANXA2 (Tyr24) in the plasma membrane area, which induced the activation of ERK1/2 to promote proliferation. These findings provide new insights into the biological functions of LncNFYB on modification of FLS, which may be exploited for the therapy of RA.

A functional variant of CD40 modulates clearance of hepatitis B virus in hepatocytes via regulation of the ANXA2/CD40/BST2 axis.

More than 250 million people in the world are chronically infected with hepatitis B virus (HBV), which causes serious complications. Host genetic susceptibility is essential for chronic hepatitis B (CHB), and our previous genome-wide association study identified a single-nucleotide polymorphism (SNP), rs1883832, in the 5' untranslated region of CD40 predisposing to chronic HBV infection, but the underlying mechanism remains undefined. This study aimed to investigate whether rs1883832 was the real functional SNP (fSNP) of CD40 and how it modulated HBV clearance in hepatocytes. We determined the fSNP of CD40 and its regulatory protein(s) using luciferase reporter assays, electrophoretic mobility shift assay, flanking restriction enhanced pulldown and chromatin immunoprecipitation. The potential anti-HBV activity of CD40 and its downstream molecule BST2 was assessed in HBV-transfected and HBV-infected hepatoma cells and HBV-infected primary human hepatocytes. Moreover, the mechanism of CD40 was investigated by mRNA sequencing, quantitative real-time polymerase chain reaction, immunofluorescence and western blot. We revealed rs1883832 as the true fSNP of CD40 and identified ANXA2 as a negative regulatory protein that preferentially bound to the risk allele T of rs1883832 and hence reduced CD40 expression. Furthermore, CD40 suppressed HBV replication and transcription in hepatocytes via activating the JAK-STAT pathway. BST2 was identified to be the key IFN-stimulated gene regulated by CD40 after activating JAK-STAT pathway. Inhibition of JAK/STAT/BST2 axis attenuated CD40-induced antiviral effect. In conclusion, a functional variant of CD40 modulates HBV clearance via regulation of the ANXA2/CD40/BST2 axis, which may shed new light on HBV personalized therapy.

Structures of annexin A2-PS DNA complexes show dominance of hydrophobic interactions in phosphorothioate binding.

The introduction of phosphorothioate (PS) linkages to the backbone of therapeutic nucleic acids substantially increases their stability and potency. It also affects their interactions with cellular proteins, but the molecular mechanisms that underlie this effect are poorly understood. Here, we report structural and biochemical studies of interactions between annexin A2, a protein that does not possess any known canonical DNA binding domains, and phosphorothioate-modified antisense oligonucleotides. We show that a unique mode of hydrophobic interactions between a sulfur atom of the phosphorothioate group and lysine and arginine residues account for the enhanced affinity of modified nucleic acid for the protein. Our results demonstrate that this mechanism of interaction is observed not only for nucleic acid-binding proteins but can also account for the association of PS oligonucleotides with other proteins. Using the anomalous diffraction of sulfur, we showed that preference for phosphorothioate stereoisomers is determined by the hydrophobic environment around the PS linkage that comes not only from protein but also from additional structural features within the ASO such as 5-Me groups on cytosine nucleobases.

Modulation of prostaglandin transport activity of SLCO2A1 by annexin A2 and S100A10.

Solute carrier organic anion transporter family member 2A1 (SLCO2A1) is a prostaglandin (PG) transporter and serves as the osmosensitive ATP-permeable maxi-anion channel (Maxi-Cl). Since a heterotetrameric complex of annexin A2 (ANXA2) and S100A10 is obligatory for the channel activity, the present study aimed to determine if they regulate SLCO2A1-mediated PG transport. This study examined PGE2 uptake and ATP release in Anxa2 and/or S100a10 knockout (KO) murine breast C127 cells. Deletion of Slco2a1 decreased PGE2-d4 uptake by wild-type (WT) cells in an isotonic medium (290 mosmol/kgH2O). Decreased osmolarity (135 mosmol/kgH2O) stimulated ATP release but did not affect PGE2 uptake kinetics, showing Km (1,280 nM) and Vmax (10.38 pmol/15 s/mg protein) similar to those in isotonic medium (1,227 nM and 10.65 pmol/15 s/mg protein), respectively, in WT cells. Deletion of Anxa2 associated with loss of S100a10 diminished SLCO2A1-mediated ATP release and uncompetitively inhibited PGE2 uptake with lowered Km (376 nM) and Vmax (2.59 pmol/15 s/mg protein). Moreover, the immunoprecipitation assay confirmed the physical interaction of ANXA2 with SLCO2A1 in WT cells. Enforcement of ANXA2 expression to Anxa2 KO cells partially restored PGE2 uptake and increased Km (744.3 nM) and Vmax (9.07 pmol/15 s/mg protein), whereas the uptake clearance (Vmax/Km) did not change much regardless of ANXA2 expression. These results suggest that an ANXA2/S100A10 complex modulates PG transport activity but osmolality has little effect on it; therefore, the bound form of SLCO2A1, which functions as a PG transporter and Maxi-Cl, may exist regardless of changes in the cell volume.NEW & NOTEWORTHY A previous study indicated that the ANXA2/S100A10 complex represents the regulatory component of SLCO2A1-mediated Maxi-Cl channel activity. The present study showed that apparent PGE2 uptake by C127 cells was osmoinsensitive and uncompetitively inhibited by loss of ANXA2 expression, demonstrating that ANXA2 is a regulatory factor of SLCO2A1-mediated PG transport activity.

FAXC interacts with ANXA2 and SRC in mitochondria and promotes tumorigenesis in cholangiocarcinoma.

Cholangiocarcinoma (CCA) is one of the most difficult malignancies to treat as the therapeutic options are limited. Although several driver genes have been identified, most remain unknown. In this study, we identified a failed axon connection homolog (FAXC), whose function is unknown in mammals, by analyzing serially passaged CCA xenograft models. Knockdown of FAXC reduced subcutaneous tumorigenicity in mice. FAXC was bound to annexin A2 (ANXA2) and c-SRC, which are tumor-promoting genes. The FAXC/ANXA2/c-SRC complex forms in the mitochondria. FAXC enhances SRC-dependent ANXA2 phosphorylation at tyrosine-24, and the C-terminal amino acid residues (351-375) of FAXC are required for ANXA2 phosphorylation. Transcriptome data from a xenografted CCA cell line revealed that FAXC correlated with epithelial-mesenchymal transition, hypoxia, and KRAS signaling genes. Collectively, these findings advance our understanding of CCA tumorigenesis and provide candidate therapeutic targets.

Annexin A2 and Ahnak control cortical NuMA-dynein localization and mitotic spindle orientation.

Proper mitotic spindle orientation depends on the correct anchorage of astral microtubules to the cortex. It relies on the remodeling of the cell cortex, a process not fully understood. Annexin A2 (Anx2; also known as ANXA2) is a protein known to be involved in cortical domain remodeling. Here, we report that in HeLa cell early mitosis, Anx2 recruits the scaffold protein Ahnak at the cell cortex facing spindle poles, and the distribution of both proteins is controlled by cell adhesion. Depletion of either protein or impaired cortical Ahnak localization result in delayed anaphase onset and unstable spindle anchoring, which leads to altered spindle orientation. We find that Ahnak is present in a complex with dynein-dynactin. Furthermore, Ahnak and Anx2 are required for correct dynein and NuMA (also known as NUMA1) cortical localization and dynamics. We propose that the Ahnak-Anx2 complex influences the cortical organization of the astral microtubule-anchoring complex, and thereby mitotic spindle positioning in human cells. This article has an associated First Person interview with the first author of the paper.

Pseudorabies Virus Regulates the Extracellular Translocation of Annexin A2 To Promote Its Proliferation.

Pseudorabies virus (PRV) infection causes enormous economic losses to the pork industry and severe health consequences in many hosts. Annexin A2 (ANXA2) is a membrane-associated protein with various intracellular functions associated with many viral infections. However, the role of ANXA2 in alphaherpesvirus replication is still not explored. In the present study, we identified the interaction between ANXA2 and PRV US3. The deficiency of ANXA2 significantly restricted PRV proliferation. PRV infection or US3 overexpression led to ANXA2 extracellular translocation. Furthermore, we confirmed that PRV or US3 could lead to the phosphorylation of the Tyr23 ANXA2 and Tyr419 Src kinase, which was associated with the ANXA2 cell surface transposition. US3 can also bind to Src in an ANXA2-independent manner and enhance the interaction between Src and ANXA2. Additionally, inhibitors targeting ANXA2 (A2ti-1) or Src (PP2) could remarkably inhibit PRV propagation in vitro and protect mice from PRV infection in vivo. Collectively, our findings broaden our understanding of the molecular mechanisms of ANXA2 in alphaherpesvirus pathogenicity and suggest that ANXA2 is a potential therapeutic target for treating alphaherpesvirus-induced infectious diseases. IMPORTANCE PRV belongs to the alphaherpesvirus and has recently re-emerged in China, causing severe economic losses. Recent studies also indicate that PRV may pose a potential public health challenge. ANXA2 is a multifunctional calcium- and lipid-binding protein implicated in immune function, multiple human diseases, and viral infection. Herein, we found that ANXA2 was essential to PRV efficient proliferation. PRV infection resulted in the extracellular translocation of ANXA2 through phosphorylation of ANXA2 and Src. ANXA2 and Src formed a complex with PRV US3. Importantly, inhibitors targeting ANXA2 or Src prevented PRV infection in vitro and in vivo. Therefore, our studies reveal a novel strategy by which alphaherpesvirus modifies ANXA2 to promote its replication and highlight ANXA2 as a target in developing novel promising antivirus agents in viral therapy.

Annexin A2 modulates phospholipid membrane composition upstream of Arp2 to control angiogenic sprout initiation.

The intersection of protein and lipid biology is of growing importance for understanding how cells address structural challenges during adhesion and migration. While protein complexes engaged with the cytoskeleton play a vital role, support from the phospholipid membrane is crucial for directing localization and assembly of key protein complexes. During angiogenesis, dramatic cellular remodeling is necessary for endothelial cells to shift from a stable monolayer to invasive structures. However, the molecular dynamics between lipids and proteins during endothelial invasion are not defined. Here, we utilized cell culture, immunofluorescence, and lipidomic analyses to identify a novel role for the membrane binding protein Annexin A2 (ANXA2) in modulating the composition of specific membrane lipids necessary for cortical F-actin organization and adherens junction stabilization. In the absence of ANXA2, there is disorganized cortical F-actin, reduced junctional Arp2, excess sprout initiation, and ultimately failed sprout maturation. Furthermore, we observed reduced filipin III labeling of membrane cholesterol in cells with reduced ANXA2, suggesting there is an alteration in phospholipid membrane dynamics. Lipidomic analyses revealed that 42 lipid species were altered with loss of ANXA2, including an accumulation of phosphatidylcholine (16:0_16:0). We found that supplementation of phosphatidylcholine (16:0_16:0) in wild-type endothelial cells mimicked the ANXA2 knock-down phenotype, indicating that ANXA2 regulated the phospholipid membrane upstream of Arp2 recruitment and organization of cortical F-actin. Altogether, these data indicate a novel role for ANXA2 in coordinating events at endothelial junctions needed to initiate sprouting and show that proper lipid modulation is a critical component of these events.

Plasminogen Receptors Promote Lipoprotein(a) Uptake by Enhancing Surface Binding and Facilitating Macropinocytosis.

BACKGROUND: High levels of Lp(a) (lipoprotein(a)) are associated with multiple forms of cardiovascular disease. Lp(a) consists of an apoB100-containing particle attached to the plasminogen homologue apo(a). The pathways for Lp(a) clearance are not well understood. We previously discovered that the plasminogen receptor PlgRKT (plasminogen receptor with a C-terminal lysine) promoted Lp(a) uptake in liver cells. Here, we aimed to further define the role of PlgRKT and to investigate the role of 2 other plasminogen receptors, annexin A2 and S100A10 (S100 calcium-binding protein A10) in the endocytosis of Lp(a). METHODS: Human hepatocellular carcinoma (HepG2) cells and haploid human fibroblast-like (HAP1) cells were used for overexpression and knockout of plasminogen receptors. The uptake of Lp(a), LDL (low-density lipoprotein), apo(a), and endocytic cargos was visualized and quantified by confocal microscopy and Western blotting. RESULTS: The uptake of both Lp(a) and apo(a), but not LDL, was significantly increased in HepG2 and HAP1 cells overexpressing PlgRKT, annexin A2, or S100A10. Conversely, Lp(a) and apo(a), but not LDL, uptake was significantly reduced in HAP1 cells in which PlgRKT and S100A10 were knocked out. Surface binding studies in HepG2 cells showed that overexpression of PlgRKT, but not annexin A2 or S100A10, increased Lp(a) and apo(a) plasma membrane binding. Annexin A2 and S100A10, on the other hand, appeared to regulate macropinocytosis with both proteins significantly increasing the uptake of the macropinocytosis marker dextran when overexpressed in HepG2 and HAP1 cells and knockout of S100A10 significantly reducing dextran uptake. Bringing these observations together, we tested the effect of a PI3K (phosphoinositide-3-kinase) inhibitor, known to inhibit macropinocytosis, on Lp(a) uptake. Results showed a concentration-dependent reduction confirming that Lp(a) uptake was indeed mediated by macropinocytosis. CONCLUSIONS: These findings uncover a novel pathway for Lp(a) endocytosis involving multiple plasminogen receptors that enhance surface binding and stimulate macropinocytosis of Lp(a). Although the findings were produced in cell culture models that have limitations, they could have clinical relevance since drugs that inhibit macropinocytosis are in clinical use, that is, the PI3K inhibitors for cancer therapy and some antidepressant compounds.

Identification and functional characterization of annexin A2 in half-smooth tongue sole (Cynoglossus semilaevis).

Annexin A2 (AnxA2), belonging to the annexin family, plays a crucial role in immune responses. In this study, the cDNA of the AnxA2 gene was identified in half-smooth tongue sole, Cynoglossus semilaevis. The transcript of AnxA2 gene in C. semilaevis (CsAnxA2) showed broad tissue distribution, with the highest expression level observed in the gut. CsAnxA2 expression was significantly up-regulated in the intestine, spleen, and kidney tissues following exposure to Shewanella algae. Immunohistochemical staining revealed that CsAnxA2 was predominantly expressed in epithelial cells and significantly elevated after S. algae challenge. Subcellular localization showed that CsAnxA2 was primarily localized in the cytoplasmic compartment. Moreover, proinflammatory cytokines (IL-6, IL-8 and IL-1ß) exhibited significant upregulation after CsAnxA2 was overexpressed in vivo. One hundred and fifty-eight CsAnxA2-interacting proteins were captured in the intestinal tissue, showing the top two normalized abundance observed for actin beta (ACTB) and protein S100-A10 (p11). Fifty-four high abundance CsAnxA2-interacting proteins (HIPs) were primary enriched in ten pathways, with the top three significantly enriched pathways being Salmonella infection, glycolysis/gluconeogenesis, and peroxisome proliferator-activated receptor (PPAR) signaling pathway. These results provide valuable information for further investigation into the functional mechanism of AnxA2 in C. semilaevis.

Upregulation of HDAC9 in hippocampal neurons mediates depression-like behaviours by inhibiting ANXA2 degradation.

Major depressive disorder (MDD) is a pervasive and devastating mental disease. Broad spectrum histone deacetylase (HDAC) inhibitors are considered to have potential for the treatment of depressive phenotype in mice. However, due to its non-specific inhibition, it has extensive side effects and can not be used in clinical treatment of MDD. Therefore, finding specific HDAC subtypes that play a major role in the etiology of MDD is the key to develop corresponding specific inhibitors as antidepressants in the future. Copy number variation in HDAC9 gene is thought to be associated with the etiology of some psychiatric disorders. Herein, we found that HDAC9 was highly expressed in the hippocampus of chronic restraint stress (CRS) mouse model of depression. Upregulation of HDAC9 expression in hippocampal neurons of mice induced depression-like phenotypes, including anhedonia, helplessness, decreased dendritic spine density, and neuronal hypoexcitability. Moreover, knockdown or knockout of HDAC9 in hippocampal neurons alleviated depression-like phenotypes caused by chronic restraint stress (CRS) in WT mice. Importantly, using immunoprecipitation-mass spectrometry (IP-MS), we further found that Annexin A2 (ANXA2) was coupled to and deacetylated by HDAC9. This coupling resulted in the inhibition of ubiquitinated ANXA2 degradation and then mediates depression-like behavior. Overall, we discovered a previously unrecognized role for HDAC9 in hippocampal neurons in the pathogenesis of depression, indicating that inhibition of HDAC9 might be a promising clinical strategy for the treatment of depressive disorders.

Prognostic significance of annexin A2 and tumor associated macrophages (TAMs) in metastatic renal cell carcinoma and their relation to Sunitinib resistance.

Sunitinib, an antiangiogenic tyrosine kinase inhibitor, is the main treatment for metastatic renal cell carcinoma (mRCC). Development of resistance is a major obstacle against therapy success. The aim of this study was to assess annexin A2 and CD163+ tumor associated macrophages (TAMs) immunohistochemical expression in 50 mRCC cases as regard to patients' prognosis and Sunitinib response. Also, to assess the correlation between annexin A2 and TAMs expression. High annexin A2 expression and TAMs density were associated with serum calcium level (P = 0.024 and 0.037, respectively), larger tumor size (P < 0.001), high tumor grade (P = 0.014 and <0.001, respectively), and the presence of tumor necrosis (P < 0.001). High annexin A2 and TAMs expressions were related to shorter patients' overall survival (P = 0.009 and 0.001, respectively) and progression-free survival (P = 0.003 and 0.001, respectively). Annexin A2 was correlated with TAMs density (r = 0.890). Annexin A2 and TAMs are associated with poor prognostic parameters in mRCC patients, including high nuclear grade, increased tumor size, and the presence of tumor necrosis, together with shorter patients' survivals and poor response to Sunitinib. Annexin A2 expression is correlated with TAMs density suggesting immunomodulatory role of annexin A2.

Catch Bond-Mediated Adhesion Drives Staphylococcus aureus Host Cell Invasion.

Various viruses and pathogenic bacteria interact with annexin A2 to invade mammalian cells. Here, we show that Staphylococcus aureus engages in extremely strong catch bonds for host cell invasion. By means of single-molecule atomic force microscopy, we find that bacterial surface-located clumping factors bind annexin A2 with extraordinary strength, indicating that these bonds are extremely resilient to mechanical tension. By determining the lifetimes of the complexes under increasing mechanical stress, we demonstrate that the adhesins form catch bonds with their ligand that are capable to sustain forces of 1500-1700 pN. The force-dependent adhesion mechanism identified here provides a molecular framework to explain how S. aureus pathogens tightly attach to host cells during invasion and shows promise for the design of new therapeutics against intracellular S. aureus.

Ca2+ ions facilitate the organization of the Annexin A2/S100A10 heterotetramer.

Annexin A2 (A2) is a member of the Annexin family, which contains Ca2+ -regulated phospholipid-binding proteins. Annexins associate with S100 proteins to form heterotetramers. The A2/S100A10 heterotetramer (A2t) is the most extensively studied of these heterotetramers. It induces membrane microdomain formation, causes membrane budding, and facilitates proliferation of some cancers. In this work, the first molecular dynamics (MD) study on the complete A2t of 868 amino acids was performed. MD trajectories of more than 600 ns each were generated for complete A2t complexes with and without Ca2+ ions. The outward extension of membrane-binding residues A2-K279 and A2-K281 was shown to be inhibited in the absence of Ca2+ as they were captured by Ca2+ -binding residue D322. F-actin binding residue A2-D339 was observed to occupy either an exposed or buried state in the absence of Ca2+ , while it only occupied the buried state in the presence of Ca2+ . The observed motions of the A2t subunits are highly organized with a strongly correlated central region which is negatively correlated with the periphery of the complex. The central region contains the S100A10 (p11) dimer, A2-N, and A2-I, while the periphery contains A2-II, A2-III, and A2-IV. Novel interactions between A2 and p11 were identified. A2 residues outside of A2-N (K80, R77, E82, and R145) had strong interactions with p11. Residue R145 of A2 may have a significant effect on the dynamics of the system, with its interaction resulting in asymmetric motions of A2. The presented results provide novel insights to inform future experimental studies.

ANXA2 as a novel substrate of FBXW7 promoting esophageal squamous cell carcinoma via ERK phosphorylation.

Our recent study suggests that FBXW7 loss of function plays a critical function in esophageal cancer. However, the mechanism of FBXW7 in promoting esophageal cancer is still unclear. Here, we explored the interaction protein of FBXW7 by screening of GST-pulldown and LC-MS/MS analysis in esophageal squamous cell carcinoma (ESCC) and identified ANXA2 as a potential target of FBXW7. FBXW7 loss of function could restore the expression of ANXA2 and promote the malignant biological characteristics of ESCC cells in vitro. Up-regulation of ANXA2 enhances the ERK pathway in ESCC. Furthermore, the 23rd tyrosine residue of ANXA2, phosphorylated by SRC, was regarded as playing important roles in the FBXW7-related degradation system. In clinical samples, we found that ANXA2 had high expression in ESCC tissues. High ANXA2 was associated with poor tumor staging. More importantly, we designed a combination regimen including SCH779284, a clinical ERK inhibitor against the phosphorylation of EKR and siRNA targeting ANXA2 by intratumor injection, and it produced potent inhibitory effects on the growth of xenograft tumors in vivo. In conclusion, this study provided evidence that FBXW7 loss of function could promote esophageal cancer through ANXA2 overexpression, and this novel regulation pathway may be used as an efficient target for ESCC treatment.

Runx1 promotes scar deposition and inhibits myocardial proliferation and survival during zebrafish heart regeneration.

Runx1 is a transcription factor that plays a key role in determining the proliferative and differential state of multiple cell types, during both development and adulthood. Here, we report how Runx1 is specifically upregulated at the injury site during zebrafish heart regeneration, and that absence of runx1 results in increased myocardial survival and proliferation, and overall heart regeneration, accompanied by decreased fibrosis. Using single cell sequencing, we found that the wild-type injury site consists of Runx1-positive endocardial cells and thrombocytes that induce expression of smooth muscle and collagen genes. Both these populations cannot be identified in runx1 mutant wounds that contain less collagen and fibrin. The reduction in fibrin in the mutant is further explained by reduced myofibroblast formation and upregulation of components of the fibrin degradation pathway, including plasminogen receptor annexin 2A as well as downregulation of plasminogen activator inhibitor serpine1 in myocardium and endocardium, resulting in increased levels of plasminogen. Our findings suggest that Runx1 controls the regenerative response of multiple cardiac cell types and that targeting Runx1 is a novel therapeutic strategy for inducing endogenous heart repair.

Reduced expression of annexin A2 is associated with impaired cell surface fibrinolysis and venous thromboembolism.

Reduced plasma fibrinolysis has been identified as a potential risk factor for venous thromboembolism (VTE), but the role of cell surface fibrinolysis in VTE is unknown. The annexin A2/S100A10 complex serves as a coreceptor for plasminogen and tissue plasminogen activator (tPA), augmenting plasmin generation by 60-fold on the endothelial cell surface. Several studies in both mice and humans support the concept that A2 regulates fibrin homeostasis and intravascular thrombosis in vivo. Here, we examined A2 protein expression and function in 115 adult subjects with VTE and 87 healthy controls. Using peripheral blood mononuclear cells as a surrogate for endothelial cells, we found a 41% mean decrease in cell surface tPA-dependent fibrinolytic activity in subjects who had a positive personal and family history of VTE but tested negative for known inherited thrombophilias (ITs). A2 protein was reduced on average by 70% and messenger RNA levels by 30%, but neither decrease correlated with anticoagulant therapy. Neither cell A2 protein nor cell surface plasmin generation correlated with plasma-based clot lysis times, suggesting that the plasma and cell surface fibrinolytic systems operate independently of one another. These data suggest that reduced expression of annexin A2 protein is associated with cell surface hypofibrinolysis and may represent a novel risk factor for IT.