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Hepatocellular carcinoma (HCC) is a highly lethal liver cancer with late diagnosis; therefore, the identification of new early biomarkers could help reduce mortality. We determine the tissue and plasma status of five annexins during hepatocarcinogenesis by diethylnitrosamine-induced cirrhosis-HCC. We found that Anxa5 was the earliest upregulated gene at week 12 after HCC initiation, while Anxa1 and Anxa2 were upregulated in advanced HCC stages (weeks 18 and 22). Furthermore, the protein level of Annexin A1, A2, A5 and A10 was increased from the early stages. Immunofluorescence and subcellular fractionation revealed Annexin A1, A2, and A5 in the cytoplasm and nuclei of tumor cells. Notably, increased plasma levels of Annexin A5 significantly (r 2  = 0.8203) correlated with Annexin A5 levels in liver tissue from week 12 and gradually increased until week 22. Using the TCGA database, we found that the expression of ANXA2 (HR = 1.7, p  = 0.0046) and ANXA5 (HR = 1.8, p  = 0.00077) was associated with poor survival in HCC patients. In conclusion, we have identified Annexin A1 and A5 as potentially useful early biomarkers for poor prognosis in HCC patients.

Efficient cell membrane repair mechanisms are essential for maintaining membrane integrity and thus for cell life. Here we show that the Ca 2+ - and phospholipid-binding proteins annexin A4 and A6 are involved in plasma membrane repair and needed for rapid closure of micron-size holes. We demonstrate that annexin A4 binds to artificial membranes and generates curvature force initiated from free edges, whereas annexin A6 induces constriction force. In cells, plasma membrane injury and Ca 2+ influx recruit annexin A4 to the vicinity of membrane wound edges where its homo-trimerization leads to membrane curvature near the edges. We propose that curvature force is utilized together with annexin A6-mediated constriction force to pull the wound edges together for eventual fusion. We show that annexin A4 can counteract various plasma membrane disruptions including holes of several micrometers indicating that induction of curvature force around wound edges is an early key event in cell membrane repair. The role of annexins in cell membrane repair is largely undefined. Here the authors use a model lipid bilayer to show that annexin A4 induces curvature at the membrane free edge and annexin A6 induces constriction force, and find that both annexins are recruited to wound edges in cells and are required for repair.

The ability of microorganisms to produce biofuels by fermentation is adversely affected by the perturbing effects of the hydrophobic biofuel on plasma membrane structure. It is demonstrated here that heterologous expression of metazoan, calcium-dependent, membrane-binding proteins of the annexin class can reduce deleterious effects of isobutanol on Saccharomyces cerevisiae viability and complex membrane functions. Therefore, expression of annexins in industrial strains of yeast or bacteria may prove beneficial in biofuel production.

Annexins are a multigene family of calcium-dependent membrane-binding proteins that play important roles in plant cell signaling. Annexins are multifunctional proteins, and their function in plants is not comprehensively understood. Arabidopsis (Arabidopsis thaliana) annexins ANN1 and ANN2 are 64% identical in their primary structure, and both are highly expressed in seedlings. Here, we showed that ann-mutant seedlings grown in the absence of sugar show decreased primary root growth and altered columella cells in root caps; however, these mutant defects are rescued by Suc, Glc, or Fru. In seedlings grown without sugar, significant up-regulation of photosynthetic gene expression and chlorophyll accumulation was found in ann-mutant cotyledons compared to that in wild type, which indicates potential sugar starvation in the roots of ann-mutant seedlings. Unexpectedly, the overall sugar content of ann-mutant primary roots was significantly higher than that of wild-type roots when grown without sugar. To examine the diffusion of sugar along the entire root to the root tip, we examined the unloading pattern of carboxyfluorescein dye and found that post-phloem sugar transport was impaired in ann-mutant root tips compared to that in wild type. Increased levels of ROS and callose were detected in the root tips of ann-mutant seedlings grown without Suc, the latter of which would restrict plasmodesmal sugar transport to root tips. Our results indicate that ANN1 and ANN2 play an important role in post-phloem sugar transport to the root tip, which in turn indirectly influences photosynthetic rates in cotyledons. This study expands our understanding of the function of annexins in plants.

Cytosolic calcium signaling is critical for regulating downstream responses in plants encountering unfavorable environmental conditions. In a genetic screen for Arabidopsis thaliana mutants defective in stress-induced cytosolic free Ca2+ ([Ca2+]cyt) elevations, we identified the R2R3-MYB transcription factor MYB30 as a regulator of [Ca2+]cyt in response to H2O2 and heat stresses. Plants lacking MYB30 protein exhibited greater elevation of [Ca2+]cyt in response to oxidative and heat stimuli. Real-time reverse transcription–polymerase chain reaction (RT-PCR) results indicated that the expression of a number of ANNEXIN (ANN) genes, which encode Ca2+-regulated membrane-binding proteins modulating cytosolic calcium signatures, were upregulated in myb30 mutants. Further analysis showed that MYB30 bound to the promoters of ANN1 and ANN4 and repressed their expression. myb30 mutants were sensitive to methyl viologen (MV) and heat stresses. The H2O2- and heat-induced abnormal [Ca2+]cyt in myb30 was dependent on the function of ANN proteins. Moreover, the MV and heat sensitivity of myb30 was suppressed in mutants lacking ANN function or by application of LaCl3, a calcium channel blocker. These results indicate that MYB30 regulates oxidative and heat stress responses through calcium signaling, which is at least partially mediated by ANN1 and ANN4.

OsANN1 is a member of the annexin protein family in rice. The function of this protein and the mechanisms of its involvement in stress responses and stress tolerance are largely unknown. Here it is reported that OsANN1 confers abiotic stress tolerance by modulating antioxidant accumulation under abiotic stress. OsANN1-knockdown [RNA interference (RNAi)] plants were more sensitive to heat and drought stresses, whereas OsANN1-overexpression (OE) lines showed improved growth with higher expression of OsANN1 under abiotic stress. Overexpression of OsANN1 promoted SOD (superoxide dismutase) and CAT (catalase) activities, which regulate H2O2 content and redox homeostasis, suggesting the existence of a feedback mechanism between OsANN1 and H2O2 production under abiotic stress. Higher expression of OsANN1 can provide overall cellular protection against abiotic stress-induced damage, and a significant accumulation of OsANN1-green fluorescent protein (GFP) signals was found in the cytosol after heat shock treatment. OsANN1 also has calcium-binding and ATPase activities in vitro, indicating that OsANN1 has multiple functions in rice growth. Furthermore, yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays demonstrated that OsANN1 interacts with OsCDPK24. This cross-talk may provide additional layers of regulation in the abiotic stress response.

Salinity (NaCl) stress impairs plant growth and inflicts severe crop losses. In roots, increasing extracellular NaCl causes Ca2+ influx to elevate cytosolic free Ca2+ ([Ca2+]cyt) as a second messenger for adaptive signaling. Amplification of the signal involves plasma membrane reduced nicotinamide adenine dinucleotide phosphate oxidase activation, with the resultant reactive oxygen species triggering Ca2+ influx. The genetic identities of the Ca2+-permeable channels involved in generating the [Ca2+]cyt signal are unknown. Potential candidates in the model plant Arabidopsis (Arabidopsis thaliana) include annexin1 (AtANN1). Here, luminescent detection of [Ca2+]cyt showed that AtANN1 responds to high extracellular NaCl by mediating reactive oxygen species-activated Ca2+ influx across the plasma membrane of root epidermal protoplasts. Electrophysiological analysis revealed that root epidermal plasma membrane Ca2+ influx currents activated by NaCl are absent from the Atann1 loss-of-function mutant. Both adaptive signaling and salt-responsive production of secondary roots are impaired in the loss-of-function mutant, thus identifying AtANN1 as a key component of root cell adaptation to salinity.

Drought and high salinity are two major abiotic stresses that significantly affect agricultural crop productivity worldwide. Annexins are a multigene family that plays an essential role in plant stress responses and various cellular processes. Here, the AnnSp2 gene was cloned from drought-resistant wild tomato ( Solanum pennellii ) and functionally characterized in cultivated tomato. AnnSp2 protein was localized in the nucleus and had higher expression in leave, flower and fruit. It was induced by several phytohormones and some abiotic stresses. Tomato plants overexpressing AnnSp2 had increased tolerance to drought and salt stress, as determined by analysis of various physiological parameters. AnnSp2-transgenic plants were less sensitive to ABA during the seed germination and seedling stages. However, under drought stress, the ABA content significantly increased in the AnnSp2-overexpressing plants, inducing stomatal closure and reducing water loss, which underlay the plants’ enhanced stress tolerance. Furthermore, scavenging reactive oxygen species (ROS), higher total chlorophyll content, lower lipid peroxidation levels, increased peroxidase activities (including APX, CAT and SOD) and higher levels of proline were observed in AnnSp2-overexpressing plants. These results indicate that overexpression of AnnSp2 in transgenic tomato improves salt and drought tolerance through ABA synthesis and the elimination of ROS.

TAR DNA-binding protein 43 (TDP-43) is an RNA binding protein found within ribonucleoprotein granules tethered to lysosomes via annexin A11. TDP-43 protein forms inclusions in many neurodegenerative diseases including amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration with TDP-43 inclusions (FTLD–TDP) and limbic predominant age-related TDP-43 encephalopathy neuropathologic change (LATE-NC). Annexin A11 is also known to form aggregates in ALS cases with pathogenic variants in ANXA11 . Annexin A11 aggregation has not been described in sporadic ALS, FTLD–TDP or LATE-NC cases. To explore the relationship between TDP-43 and annexin A11, genetic analysis of 822 autopsy cases was performed to identify rare ANXA11 variants. In addition, an immunohistochemical study of 368 autopsy cases was performed to identify annexin A11 aggregates. Insoluble annexin A11 aggregates which colocalize with TDP-43 inclusions were present in all FTLD–TDP Type C cases. Annexin A11 inclusions were also seen in a small proportion (3–6%) of sporadic and genetic forms of FTLD–TDP types A and B, ALS, and LATE-NC. In addition, we confirm the comingling of annexin A11 and TDP-43 aggregates in an ALS case with the pathogenic ANXA11 p.G38R variant. Finally, we found abundant annexin A11 inclusions as the primary pathologic finding in a case of progressive supranuclear palsy-like frontotemporal dementia with prominent striatal vacuolization due to a novel variant, ANXA11 p.P75S. By immunoblot, FTLD–TDP with annexinopathy and ANXA11 variant cases show accumulation of insoluble ANXA11 including a truncated fragment. These results indicate that annexin A11 forms a diverse and heterogeneous range of aggregates in both sporadic and genetic forms of TDP-43 proteinopathies. In addition, the finding of a primary vacuolar annexinopathy due to ANXA11 p.P75S suggests that annexin A11 aggregation is sufficient to cause neurodegeneration.

Genetic disruption of the dystrophin complex produces muscular dystrophy characterized by a fragile muscle plasma membrane leading to excessive muscle degeneration. Two genetic modifiers of Duchenne Muscular Dystrophy implicate the transforming growth factor β (TGFβ) pathway, osteopontin encoded by the SPP1 gene and latent TGFβ binding protein 4 (LTBP4). We now evaluated the functional effect of these modifiers in the context of muscle injury and repair to elucidate their mechanisms of action. We found that excess osteopontin exacerbated sarcolemmal injury, and correspondingly, that loss of osteopontin reduced injury extent both in isolated myofibers and in muscle in vivo. We found that ablation of osteopontin was associated with reduced expression of TGFβ and TGFβ-associated pathways. We identified that increased TGFβ resulted in reduced expression of Anxa1 and Anxa6, genes encoding key components of the muscle sarcolemma resealing process. Genetic manipulation of Ltbp4 in dystrophic muscle also directly modulated sarcolemmal resealing, and Ltbp4 alleles acted in concert with Anxa6, a distinct modifier of muscular dystrophy. These data provide a model in which a feed forward loop of TGFβ and osteopontin directly impacts the capacity of muscle to recover from injury, and identifies an intersection of genetic modifiers on muscular dystrophy.