Annexin Family Of Proteins Research Articles

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.

Comprehensive analyses of annexins in naked carp (Gymnocypris przewalskii) unveil their roles in saline-alkaline stress

Naked carp (Gymnocypris przewalskii) is a migratory fish endemic to the Qinghai Lake, the largest inland saline-alkaline lake in China. Based on our previous results of comparative transcriptome sequencing, we found that pathways related to regulation of actin cytoskeleton, plasma membrane damage, and inflammation-related signaling were significantly enriched during saline-alkaline stress, suggesting involvement of annexin (ANN) family proteins in stress adaption in naked carp. Herein, to explore the role of annexin family in naked carp under saline-alkaline stress, twenty GpANNs family members were identified at the genome-wide scale, and bioinformatics and expression analysis under saline-alkaline stress were performed. The predicted subcellular localization showed that most members of GpANNs were distributed in the cytoplasm, and the rest were localized in the cytoskeleton, nucleus, or mitochondria, respectively. GpANNs family proteins contained specific type II Ca2+-binding motifs, actin-binding motifs, GTP-binding motifs, etc. Phylogenetic tree analysis showed that the annexin homologues from naked carp, carp (Cyprinus carpio), zebrafish (Danio rerio), and chinese cavefish (Sinocyclocheilus rhinocerous) were divided into eight groups, i.e. ANN1 ∼ 6, ANN11 and ANN13. But homologues of ANN7 ∼ 10 groups were not found in these three fish species. Twenty-eight, nineteen and twenty-nine orthologous gene pairs were identified between naked carp and carp, zebrafish and chinese cavefish, respectively. Six paralogous gene pairs were identified in naked carp, all of which had a Ka/Ks ratio less than one, suggesting that the GpANNs family may have undergone purifying selection pressure during evolution. Expression of GpANN3 was significantly up-regulated in the kidney tissue under saline-alkaline stress, while GpANN2 was significantly down-regulated. Similar trends of protein expression were also observed by Western blot, suggesting involvement of GpANN3 and GpANN2 in stress response. Confocal microscopy revealed that GpANN2 was localized in both cytoplasm and nucleus, but GpANN3 was preferentially localized in the cytoplasm. Our study demonstrated the role of annexins in stress adaption, and provided the basic information required for further function analyses in naked carp.

Identification of an AnnexinB9 involve in white spot syndrome virus infection in red claw crayfish Cherax quadricarinatus

Annexin (Anx) family protein is a highly conserved protein family that plays important roles in immune defense of vertebrates and invertebrates against invading pathogens. In this study, a novel Anx was cloned and characterized from the red claw crayfish, Cherax quadricarinatus. The Open Reading Frame of CqAnxB9 consisted of 930 nucleotide bases pair and encoded 309 amino acids. The CqAnxB9 protein contained three repeat Anx domains and a typical KGLGT sequence. Tissue expression analysis showed that the expression levels of CqAnxB9 were mainly expressed in the intestine, hepatopancreas and hemocytes. After WSSV challenge, CqAnxB9 expression was up-regulated in the hematopoietic tissue (Hpt) cells. Moreover, knockdown of CqAnxB9 inhibited WSSV replication and VP28 expression, suggesting that CqAnxB9 plays a positive role in WSSV infection. Further studies revealed that recombinant CqAnxB9 protein was found to bind to the viral envelop protein VP28. All these findings indicate that new-found CqAnxB9 is likely to promote WSSV infection in crustaceans, which provides a better understanding of the pathogenesis of WSSV.

Interplay of membrane crosslinking and curvature induction by annexins

Efficient plasma membrane repair (PMR) is required to repair damage sustained in the cellular life cycle. The annexin family of proteins, involved in PMR, are activated by Ca2+ influx from extracellular media at the site of injury. Mechanistic studies of the annexins have been overwhelmingly performed using a single annexin, despite the recruitment of multiple annexins to membrane damage sites in living cells. Hence, we investigate the effect of the presence of the crosslinking annexins, annexin A1, A2 and A6 (ANXA1, ANXA2 and ANXA6) on the membrane curvature induction of annexin A4 (ANXA4) in model membrane systems. Our data support a mechanistic model of PMR where ANXA4 induced membrane curvature and ANXA6 crosslinking promotes wound closure. The model now can be expanded to include ANXA1 and ANXA2 as specialist free edge membrane crosslinkers that act in concert with ANXA4 induced curvature and ANXA6 crosslinking.

Possible involvement of annexin A6 in preferential sperm penetration in the germinal disk region.

Graphical abstract During fertilization, avian sperm preferentially penetrate into the perivitelline membrane that covers the germinal disk region where the female nucleus is present. This phenomenon has been observed not only in domestic birds but also in wild birds; however, the mechanisms controlling sperm preference are still unclear. In this study, we investigated the possible involvement of annexin family protein in sperm–egg interaction in Japanese quail. Microscopic examination of fertilized eggs indicated that quail sperm penetration only occurred in the germinal disk region, and sperm localized outside the germinal disk were trapped in the perivitelline membrane. Western blot analysis and immunofluorescence microscopy revealed the presence of annexin A1 and A6 in the oocyte membrane, while annexin A6 localized in the perivitelline space of the germinal disk region. Further, our sperm binding assay using recombinant annexin A6 demonstrated that ejaculated sperm specifically bound to annexin A6 expressed in mammalian cell lines. These results suggest that annexin A6, which is expressed on the surface of oocytes, may function in sperm–egg interaction in the germinal disk region and that this binding may ensure sperm retention on the surface of the egg plasma membrane until fertilization takes place in Japanese quail.Lay summaryIn bird species, fertilization takes place immediately after ovulation of the egg. Sperm preferentially penetrate a specific area of the egg coating that covers the ‘germinal disk region’ – this area contains the cell that needs to be fertilized by a sperm. However, since the bird egg is extremely large in size and sperm must reach the ‘germinal disk region’ to achieve fertilization, it is unclear how this happens. Annexin proteins support fertilization in mammals, and we found that annexin A6 protein exhibits a unique localization in the germinal disk region in the eggs of Japanese quail. To test this interaction, we incubated quail sperm with cells that produced annexin A6 and found that ejaculated sperm bound to the cells. These results suggest that annexin A6 may have a role in the sperm–egg interaction in the germinal disk region in Japanese quail.

Analysis of the Expression and Prognostic Value of Annexin Family Proteins in Bladder Cancer.

BackgroundBladder cancer (BC) is the most common tumor of the urinary system. Non-muscle-invasive bladder cancer (NMIBC) has a high recurrence rate after surgery, and patients with muscle-invasive bladder cancer (MIBC) have poor quality of life after radical surgery. Understanding the molecular mechanism of bladder cancer is helpful for providing a more appropriate treatment approach. Annexins are calcium-binding proteins and play an important role in different tumor cells. However, the role of the annexin family in bladder cancer has not been studied in detail.MethodsONCOMINE, UALCAN, TIMER2.0, Kaplan-Meier Plotter, cBioPortal, and WebGestalt were utilized in this study.ResultsANXA2, ANXA3, ANXA4, ANXA8, and ANXA9 were significantly increased in bladder tumor tissues, while ANXA6, ANXA7, and ANXA11 were significantly decreased. ANXA1, ANXA2, ANXA3, ANXA5, ANXA6, ANXA7, and ANXA9 had prognostic value in bladder cancer. In addition, specific annexins were specifically expressed in different subtypes of MIBC and were related to the histological morphology of bladder tumors. ANXA1, ANXA2, ANXA3, ANXA5, ANXA6, ANXA7, and ANXA8 were highly expressed in basal-subtype MIBC, while ANXA4, ANXA9, ANXA10, and ANXA11 were mainly expressed in luminal-subtype MIBC. Finally, we analyzed the possible mechanisms of ANXAs in different subtypes of bladder cancer through GO and KEGG analyses and the correlation between ANXAs and immune infiltration in the tumor microenvironment.ConclusionTaken together, our results indicate that annexins might play important roles in BC and have the potential to be used as markers for subtype classification.

Annexins A1 and A2 Accumulate and Are Immobilized at Cross-Linked Membrane-Membrane Interfaces.

Rapid membrane repair is required to ensure cell survival after rupture of the plasma membrane. The annexin family of proteins is involved in plasma membrane repair (PMR) and is activated by the influx of Ca2+ from the extracellular medium at the site of injury. Annexins A1 and A2 (ANXA1 and ANXA2, respectively) are structurally similar and bind to negatively charged phosphatidylserine (PS) to induce membrane cross-linking and to promote fusion, which are both essential processes that occur during membrane repair. The degree of annexin accumulation and the annexin mobility at cross-linked membranes are important aspects of ANXA1 and ANXA2 function in repair. Here, we quantify ANXA1- and ANXA2-induced membrane cross-linking between giant unilamellar vesicles (GUVs). Time-lapse measurements show that ANXA1 and ANXA2 can induce membrane cross-linking on a time scale compatible with PMR. Cross-linked membrane-membrane interfaces between the GUVs persist in time without fusion, and quantification of confocal microscopy images demonstrates that ANXA1, ANXA2, and, to a lesser extent, PS lipids accumulate at the double membrane interface. Fluorescence recovery after photobleaching shows that the annexins are fully immobilized at the double membrane interface, whereas PS lipids display a 75% decrease in mobility. In addition, the complete immobilization of annexins between two membranes indicates a high degree of network formation between annexins, suggesting that membrane cross-linking is mainly driven by protein-protein interactions.

Molecular characterization and expression analysis of annexin B3 and B38 as secretory proteins in Echinococcus granulosus

BackgroundCystic echinococcosis is a parasitic zoonotic disease, which poses a threat to public health and animal husbandry, and causes significant economic losses. Annexins are a family of phospholipid-binding proteins with calcium ion-binding activity, which have many functions.MethodsTwo annexin protein family genes [Echinococcus granulosus annexin B3 (EgAnxB3) and EgAnxB38] were cloned and molecularly characterized using bioinformatic analysis. The immunoreactivity of recombinant EgAnxB3 (rEgAnxB3) and rEgAnxB38 was investigated using western blotting. The distribution of EgAnxB3 and EgAnxB38 in protoscoleces (PSCs), the germinal layer, 18-day strobilated worms and 45-day adult worms was analyzed by immunofluorescence localization, and their secretory characteristics were analyzed preliminarily; in addition, quantitative real-time reverse transcription polymerase chain reaction was used to analyze their transcript levels in PSCs and 28-day strobilated worms stages. The phospholipid-binding activities of rEgAnxB3 and rEgAnxB38 were also analyzed.ResultsEgAnxB3 and EgAnxB38 are conserved and contain calcium-binding sites. Both rEgAnxB3 and rEgAnxB38 could be specifically recognized by the serum samples from E. granulosus-infected sheep, indicating that they had strong immunoreactivity. EgAnxB3 and EgAnxB38 were distributed in all stages of E. granulosus, and their transcript levels were high in the 28-day strobilated worms. They were found in liver tissues near the cysts. In addition, rEgAnxB3 has Ca2+-dependent phospholipid-binding properties.ConclusionsEgAnxB3 and EgAnxB38 contain calcium-binding sites, and rEgAnxB3 has Ca2+-dependent phospholipid-binding properties. EgAnxB3 and EgAnxB38 were transcribed in PSCs and 28-day strobilated worms. They were expressed in all stages of E. granulosus, and distributed in the liver tissues near the hydatid cyst, indicating that they are secreted proteins that play a crucial role in the development of E. granulosus.Graphical

Investigating Plasma-Membrane Repair Employing Thermoplasmonics

Integrity of the cell's plasma membrane (PM) is paramount, not only for the survival of healthy cells, but it also plays a pivotal role in the development and spreading of cancer. The annexin protein family is largely involved in the repair machinery of the cell's PM. Importantly, highly invasive cancers like lymphoma and central nervous system cancers have an upregulated annexin V (ANXA5) expression, while others like Leukemia show a strong downregulations of ANXA5. These observations suggest an important role of annexins in these type of cellular disorders.

Zone of Interaction Between the Parasite and the Host: Protein Profile of the Body Cavity Fluid of Gasterosteus aculeatus L. Infected with the Cestode Schistocephalus solidus (Muller, 1776).

During infection, the host and the parasite "communicate" with each other through various molecules, including proteins. The aim of this study was to describe the excretory-secretory proteins from the helminth Schistocephalus solidus and its intermediate host, the three-spined stickleback Gasterosteus aculeatus L., which are likely to be involved in interactions between them. Combined samples of washes from the G. aculeatus sticklebacks cavity infected with the S. solidus, and washes from the parasite surface were used as experimental samples, while washes from the uninfected fish body cavity were used as control. The obtained samples were analyzed using mass-spectrometry nLC-MS/MS. As a result of mass-spectrometry analysis 215 proteins were identified. Comparative quantitative analysis revealed significant differences in LFQ intensity between experimental and control samples for 20 stickleback proteins. In the experimental samples, we found an increase in the content of serpins, plasminogen, angiotensin 1-10, complement component C9, and a decrease in the content of triosephosphate isomerase, creatine kinase, fructose-biphosphate aldolase, superoxide dismutase, peroxidoxin-1, homocysteine-binding and fatty acid-binding proteins, compared to uninfected fish samples. In the experimental group washes, 30 S. solidus proteins were found, including malate dehydrogenase, annexin family proteins, serpins, peptidyl-prolyl cis-trans isomerase and fatty acid-binding protein. Thus, the protein composition of washes from the helminth S. solidus surface and the body cavity of infected and uninfected stickleback G. aculeatus were studied. As a result, it was shown that various components of the immune defense system predominated in the washes of infected fish and helminths.

Annexin A3 upregulates the infiltrated neutrophil-lymphocyte ratio to remodel the immune microenvironment in hepatocellular carcinoma

Accumulating evidence has indicated that inflammation is required for the initiation and progression of hepatocellular carcinoma (HCC). The annexin family protein, which has a highly similar structure, has been demonstrated to participate in pro- or anti-inflammatory regulation in the developing of tumours. However, the potential effects of ANXA3 in the immune microenvironment of HCC remain unknown. In present study, we found that increased ANXA3 expression is associated with a higher infiltrated neutrophil-lymphocyte ratio (iNLR) in HCC. Moreover, HCC patients with a high iNLR and high ANXA3 expression confer the highest risk of death. ANXA3 can be detected in both cell lysates and culture supernatants. However, the secretory ANXA3 did not directly regulate the iNLR. Further study demonstrated that ANXA3 upregulated the iNLR by inducing chemokine CXCL8 and CCL25 release from HCC cells. We further confirmed that ANXA3 promotes tumourigenesis and detected the same associations between ANXA3 and the iNLR or chemokines in vivo. Our findings indicate that ANXA3 regulates the chemokine to remodel the iNLR and promotes tumourigenicity in HCC. These results further expanded our understanding of ANXA3 in the microenvironment of HCC and might provide novel targets for the investigation of molecular treatments for HCC patients.

33-kDa ANXA3 isoform contributes to hepatocarcinogenesis via modulating ERK, PI3K/Akt-HIF and intrinsic apoptosis pathways

IntroductionAs a member of annexin family proteins, annexin A3 (ANXA3) has 36-kDa and 33-kDa isoforms. ANXA3 plays crucial roles in the tumorigenesis, aggressiveness and drug-resistance of cancers. However, previous studies mainly focused on the role of total ANXA3 in cancers without distinguishing the distinction between the two isoforms, the role of 33-kDa ANXA3 in cancer remains unclear. ObjectivesCurrent work aimed to investigate the function and regulation mechanism of 33-kDa ANXA3 in hepatocarcinoma. MethodsThe expressions of ANXA3, CRKL, Rac1, c-Myc and pAkt were analyzed in hepatocarcinoma specimens by Western blotting. The biological function of 33-kDa ANXA3 in the growth, metastasis, apoptosis, angiogenesis, chemoresistance of hepatocarcinoma cells with the underlying molecular mechanism were investigated using gain-of-function strategy in vitro or in vivo. Results33-kDa ANXA3 was remarkably upregulated in tumor tissues compared with corresponding normal liver tissues of hepatocarcinoma patients. Its stable knockdown decreased the in vivo tumor growing velocity and malignancy of hepatocarcinoma HepG2 cells transplanted in nude mice. The in vitro experimental results indicated 33-kDa ANXA3 knockdown suppressed the proliferation, colony forming, migration and invasion abilities of HepG2 cells through downregulating CRKL, Rap1b, Rac1, pMEK, pERK2 and c-Myc in ERK pathway; inhibited angiogenesisability of HepG2 cells through inactivating PI3K/Akt-HIF pathway; induced apoptosis and enhanced chemoresistance of HepG2 cells through increasing Bax/decreasing Bcl-2 expressions and inactivating caspase 9/caspase 3 in intrinsic apoptosis pathway. Accordingly, CRKL, Rac1, c-Myc and pAkt were also upregulated in hepatocarcinoma patients ’ tumor tissues compared with corresponding normal liver tissues. ConclusionsThe overexpression of 33-kDa ANXA3 is involved in the clinical progression of hepatocarcinoma and in the malignancy, angiogenesis and apoptosis of hepatocarcinoma cells. It is of potential use in hepatocarcinoma diagnosis and treatment.

Proteomic profiling of sclerotic hippocampus revealed dysregulated packaging of vesicular neurotransmitters in temporal lobe epilepsy

PurposeTemporal lobe epilepsy (TLE) is the most common type of epilepsy. Hippocampal sclerosis is the most distinctive pathological feature of TLE; however, its role in the pathogenesis of TLE remains to be clarified. We performed global protein expression analysis of hippocampus from TLE patients and controls, aiming to reveal the molecular signaling pathways related to TLE. MethodProteomic and bioinformatic analyses of the hippocampus were performed on 4 TLE and 4 control samples. High-resolution liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS), in combination with TMT-6plex quantification, was applied for global protein expression analysis. The proteomics results were validated by Western blot with 25 TLE and 25 control individuals and Immunohistochemistry analysis with 33 TLE and 10 control individuals. ResultsBioinformatics analysis demonstrated differentially expressed proteins in the synaptic vesicle pathway, the prostaglandin synthesis and regulation pathway and endocannabinoids and retrograde modulation of synaptic transmission pathway. Among these, excitatory amino acid transporter 1 (EAAT1) and Vesicular glutamate transporter 1 (VGLUT1) are critical for TLE and dysregulated expression might be closely related to the uptake of extracellular glutamate and contribute to the pathophysiology of TLE. Ras-related protein Rab-3A (RAB3A) downregulation might indicate the TLE-induced compensatory deficit in glutamate release. ConclusionOur study indicates that expression of some proteins involved in the packaging of vesicular neurotransmitters is altered in TLE. In addition, upregulated expression of annexin family proteins, which are also related to TLE, might play an important role in protection against TLE.

Annexins Bend Wound Edges during Plasma Membrane Repair.

The plasma membrane of eukaryotic cells defines the boundary to the extracellular environment and, thus provides essential protection from the surroundings. Consequently, disruptions to the cell membrane triggered by excessive mechanical or biochemical stresses pose fatal threats to cells, which they need to cope with to survive. Eukaryotic cells cope with these threats by activating their plasma membrane repair system, which is shared by other cellular functions, and includes mechanisms to remove damaged membrane by internalization (endocytosis), shedding, reorganization of cytoskeleton and membrane fusion events to reseal the membrane. Members of the annexin protein family, which are characterized by their Ca2+-dependent binding to anionic phospholipids, are important regulators of plasma membrane repair. Recent studies based on cellular and biophysical membrane models show that they have more distinct functions in the repair response than previously assumed by regulating membrane curvature and excision of damaged membrane. In cells, plasma membrane injury and flux of Ca2+ ions into the cytoplasm trigger recruitment of annexins including annexin A4 and A6 to the membrane wound edges. Here, they induce curvature and constriction force, which help pull the wound edges together for eventual fusion. Cancer cells are dependent on efficient plasma membrane repair to counteract frequent stress-induced membrane injuries, which opens novel avenues to target cancer cells through their membrane repair system. Here, we discuss mechanisms of single cell wound healing implicating annexin proteins and membrane curvature.

33-kDa ANXA3 Knockdown Suppresses Carcinogenesis, Angiogenesis and Enhances Chemoresistance of Hepatocarcinoma <i>via</i> Inhibiting ERK, PI3K/Akt-HIF and Intrinsic Apoptosis Pathways

Background: As a member of annexin family proteins, annexin A3 (ANXA3) has 36-kDa and 33-kDa isoforms, which plays important roles in aggressiveness, tumorigenesis and drug resistance in tumors. Previous studies mainly focused on the role of total ANXA3 in cancers without distinguishing the distinction between the two isoforms, the role of 33-kDa ANXA3 in cancer remains unclear. Current work established the potential role and regulation mechanism of 33-kDa ANXA3 in hepatocarcinoma. Methods: The expression patterns of ANXA3, CRKL, Rac1, c-Myc and pAkt were analyzed in hepatocarcinoma specimens by western blotting; The biological function of 33-kDa ANXA3 in hepatocarcinoma cell growth, metastasis, apoptosis, angiogenesis, chemoresistance and the underlying molecular mechanism were investigated using gain-of-function strategyin vitro or in vivo. Findings: 33-kDa ANXA3 protein was significantly upregulated in cancerous tissues compared with paracancerous non-tumor liver tissues of hepatocarcinoma patients. Its stable knockdown decreased the in vivo tumor growing velocity and malignancy of hepatocarcinoma HepG2 cells transplanted in nude mice. The in vitro experimental results indicated 33-kDa ANXA3 knockdown suppressed proliferative, colony forming, migrative and invasive abilities of HepG2 cells through downregulating CRKL, Rap1b, Rac1, pMEK, pERK2 and c-Myc in ERK pathway; inhibited angiogenesis ability of HepG2 cells through inactivating PI3K/Akt-HIF pathway; induced apoptosis and enchanced chemoresistance of HepG2 cells through increasing Bax/decreasing Bcl-2 expressions and inactivating caspase 9/caspase 3 in intrinsic apoptosis pathway. Accordingly, CRKL, Rac1, c-Myc and pAkt were also upregulated in cancerous tissues compared with paracancerous non-tumor liver tissues of hepatocarcinoma patients. Interpretation: 33-kDa ANXA3 plays vital role in hepatocarcinoma malignancy, angiogenesis and chemoresistance. It is of potential use in prognosis and therapeutics for hepatocarcinoma. Funding Statement: This work was supported by grants from National Natural Science Foundation of China (81672737, 81272186, 31900517) and Natural Science Foundation of Liaoning (LZ2019003, 20181550168, LQ2017001). Declaration of Interests: The authors declare no potential conflicts of interest. Ethics Approval Statement: Tissue use and study protocol were approved by the Medical Ethics Committee of Dalian Medical University. Informed consent was obtained from each patient. All experiment methods were performed in accordance with the relevant guidelines and regulations.

Abstract 107: Extracellular vesicles from cancer associated fibroblasts induce drug resistance via integrinβ1/FAK signaling in gastric cancer cells

Abstract Background: Cancer-associated fibroblasts (CAFs) enhance tumor progression through secretion of soluble factors. Although we demonstrated that direct-interaction between CAFs and cancer cells confer on gastric cancer (GC) cells the ability to invade extracellular matrix (ECM), the molecular mechanism underlying the drug resistance by soluble factors from CAFs has not been determined. Methods: We have established primary fibroblasts from more than 100 GC patients and collected conditioned medium (CM) of CAFs. Human GC cell lines cultured in normal medium or CAF-CM were examined the drug resistance with or without extracellular matrix (ECM). We isolated extracellular vesicles (EVs) from CAF-CM by ultracentrifugal separation. We performed RNA sequencing of GC cell lines and mass analysis of GC-EVs and CAF-EVs. Results: GC cells treated with CAF-CM or CAF-EVs did not show morphological change on normal coated plates. Notably, we found that GC cells with CAF-CM or CAF-EVs on ECM-coated plates showed unique network formation and cisplatin resistance. Given that the morphological change appeared within 1-3 hours, we predicted that the stimulation by CAF-EV had an impact on the expression pattern of membrane proteins in GC cells. Mass analysis using isolated CAF-EVs was conducted and revealed that CAF-EVs contained high amount of annexin family protein members. Moreover, we showed that annexin in CAF-EVs is critical for network formation and drug resistance of GC cells. Finally, we identified integrinβ1/FAK signaling was activated in GC cells treated with CAF-EVs during network formation. Conclusions: These findings highlight the underlying mechanism whereby CAF-EVs in tumor-microenvironment confer GC cells on drug resistance. Citation Format: Tomoyuki Uchihara, Atsuko Yonemura, Keisuke Miyake, Tadahito Yasuda, Rumi Itoyama, Masaaki Iwatsuki, Yoshifumi Baba, Naoya Yoshida, Hideo Baba, Takatsugu Ishimoto. Extracellular vesicles from cancer associated fibroblasts induce drug resistance via integrinβ1/FAK signaling in gastric cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 107.

Regulation of sperm storage and movement in the ruminant oviduct

Three regions of the ruminant oviduct play different roles in the progress of sperm: the uterotubal junction, isthmus, and ampulla. The uterotubal junction acts as a point of selection of sperm, requiring that sperm are progressively motile and express specific proteins in order to enter the oviduct. The isthmus stores sperm, preserving motility and viability until ovulation. Sperm are stored in the isthmus by binding to its mucosal epithelium. In bovine sperm, binding to the oviductal epithelium is promoted by proteins that are secreted by the seminal vesicles and coat the heads of sperm by associating with plasma membrane phospholipids. Putative oviductal receptors for the seminal vesicle proteins are members of the annexin protein family. Release of sperm from the storage site in the isthmus is gradual, which serves to ensure that sperm in the proper physiological state reach the oocytes at the appropriate moment and also to reduce incidence of polyspermic fertilization. The ampulla supports fertilization and may participate in guiding sperm toward the eggs. Further studies are needed to improve our understanding of the interactions between sperm and the female reproductive tract, in order to develop means to improve fertility in ruminants.

ANXA5 Enhances Malignancy of Murine Hepatocarcinoma Hca-P Cells <i>via</i> ERK Activation and E-cadherin Suppression

The invasion and metastasis are linked to the rapid progression and poor prognosis of hepatocarcinoma patients. Lymphatic metastasis is potentially involved in above pathogenesis with unclear mechanism. Previously, we found the deregulation of annexin A5 (ANXA5), a member of Ca²⁺-regulated phospholipid- and membrane-binding annexin family protein, mediated the <i>in vivo</i> malignancy, lymph node metastasis (LNM) rate and level of mice transplanted with Hca-P, a murine hepatocarcinoma cell line with the LNM potential rate of ~25%. Current work aimed to investigate the influence with action mechanism of ANXA5 overexpression on the <i>in vitro</i> malignant behaviours of Hca-P cells. For overexpressing ANXA5, the <i>Anxa5</i> gene was ligated into pCDNA3.1 (+) vector and transfected into Hca-P named as Hca-P-ANXA5up. Hca-P transfected with empty pCDNA3.1 (+) vector was named as Hca-P-mock and used as the control. The monoclonal Hca-P-mock and Hca-P-ANXA5up cell lines were obtained against G418 screening using limiting dilution method. Compared with the Hca-P-mock cells, Western blotting assay indicated ANXA5 expression level was increased by 50.1% (<i>p</i>=0.025) in the Hca-P-ANXA5up cells. Transwell chamber assays indicated that the migration and invasion capacities of Hca-P-ANXA5up cells were increased by 150.2% (<i>p</i>=0.001) and 94.8% (<i>p</i>=0.003) than Hca-P-mock cells. ANXA5 overexpression enhanced the levels of p-MEK (Ser217/221), ERK1, ERK2, p-ERK1 (Thr202/Tyr204) and p-ERK2 (Thr185/Tyr187), and suppressed the levels of E-Cadherin, Snail and Slug in Hca-P cells. Current work shows ANXA5 overexpression enhances the malignant behaviours of hepatocarcinoma Hca-P cells through activating p-MEK-ERK pathway and suppressing E-Cadherin, Snail and Slug. It is of potential value in tumor malignancy and lymphatic metastasis of hepatocarcinoma.

Annexins induce curvature on free-edge membranes displaying distinct morphologies

Annexins are a family of proteins characterized by their ability to bind anionic membranes in response to Ca2+-activation. They are involved in a multitude of cellular functions including vesiculation and membrane repair. Here, we investigate the effect of nine annexins (ANXA1-ANXA7, ANXA11, ANXA13) on negatively charged double supported membrane patches with free edges. We find that annexin members can be classified according to the membrane morphology they induce and matching a dendrogam of the annexin family based on full amino acid sequences. ANXA1 and ANXA2 induce membrane folding and blebbing initiated from membrane structural defects inside patches while ANXA6 induces membrane folding originating both from defects and from the membrane edges. ANXA4 and ANXA5 induce cooperative roll-up of the membrane starting from free edges, producing large rolls. In contrast, ANXA3 and ANXA13 roll the membrane in a fragmented manner producing multiple thin rolls. In addition to rolling, ANXA7 and ANXA11 are characterized by their ability to form fluid lenses localized between the membrane leaflets. A shared feature necessary for generating these morphologies is the ability to induce membrane curvature on free edged anionic membranes. Consequently, induction of membrane curvature may be a significant property of the annexin protein family that is important for their function.

Functional Association between Regulatory RNAs and the Annexins.

Cells respond to pathophysiological states by activation of stress-induced signalling. Regulatory non-coding microRNAs (miRNAs) often form stable feed-forward loops which ensure prolongation of the signal, contributing to sustained activation. Members of the annexin protein family act as sensors for Ca2+, pH, and lipid second messengers, and regulate various signalling pathways. Recently, annexins were reported to participate in feedback loops, suppressing miRNA synthesis and attenuating stress-induced dysregulation of gene expression. They can directly or indirectly associate with RNAs, and are transferred between the cells in exosomes and shed microvesicles. The ability of annexins to recruit other proteins and miRNAs into exosomes implicates them in control of cell–cell interactions, affecting the adaptive responses and remodelling processes during disease. The studies summarized in this Review point to an emerging role of annexins in influencing the synthesis, localisation, and transfer of regulatory RNAs.