Natural Products from Leaves of the Ancient Iranian Medicinal Plant Echium amoenum Fisch. & C. A. Mey.

For several millennia, leaves of Echium amoenum Fisch. & C. A. Mey., an important Iranian medicinal plant with nutritional value as nutraceutical, have been used as tea for the treatment of several conditions, including inflammation. The nutritional value of intake of E. amoenum tea has mainly been correlated to its rich content of mainly water-soluble antioxidants. Although the entire plant is utilized, only natural products of the flowers have previously been thoroughly investigated. The rare natural products bis(3-(3,4-dihydroxyphenyl)-1-methoxy-1-oxopropan-2-yl)-1-(3,4-dihydroxyphenyl)-6,7-dihydroxy-1,2-dihydronaphthalene-2,3-dicarboxylate, 4-Oxy-(E)-caffeoyl-2,3-dihydroxybutanoic acid methyl ester and 4-Oxy-(Z)-caffeoyl-2,3-dihydroxybutanoic acid methyl ester, in addition to the widely distributed compounds rosmarinic acid methyl ester and (E)-caffeic acid, were purified and characterized from leaves of Echium amoenum. The structures were determined by a combination of several 2D NMR spectroscopic techniques, circular dichroism spectroscopy and high-resolution mass spectrometry. The fact that bis(3-(3,4-dihydroxyphenyl)-1-methoxy-1-oxopropan-2-yl)-1-(3,4-dihydroxyphenyl)-6,7-dihydroxy-1,2-dihydronaphthalene-2,3-dicarboxylate belongs to a rare group of natural products which have previously been patented for their significant anti-inflammatory activity may rationalize the traditional treatment of inflammations with E. amoenum.

The cooperative folding of annexin A2 relies on a transient nonnative intermediate.

Annexins (Anxs) are a family of highly homologous proteins that bind and aggregate lipid vesicles in the presence of calcium. All members of the family contain a variable N-terminus determining specific functions, followed by a conserved core region responsible for the general calcium-dependent lipid-binding property. The core structure consists of four homologous domains (DI-DIV), each consisting of a right-handed super-helix of five α-helices. We present data from a combination of site-directed mutagenesis, NMR, and circular dichroism showing that the G25-D34 region of the N-terminus as well as the contacts between residues D38A, R63A, and Q67A of AnxA2-DI are crucial for the autonomous folding and stability of DI of AnxA2. However, we also show that the folding of the full-length protein is very robust in that mutations and truncations that disrupted the folding of AnxA2-DI did not abolish the folding of full-length AnxA2, only lowering its thermal stability. This robustness of the folding of full-length AnxA2 is likely to be mediated by the existence of at least one transient nonnative intermediate as suggested by our kinetic data using stopped-flow fluorescence experiments. We also show that hydrophobic amino acids in AnxA2-DI involved in interfacial contacts with AnxA2-DIV are important for the cooperative folding and stability of the full-length protein. Mutating all of the V57E-V98R-G101Y residues in AnxA2-DI did not affect the folding of the domain, only its stability, but prevented the cooperative folding of the full-length protein. Our collective results favor a highly cooperative and robust folding process mediated by alternative intermediate steps. Since AnxA2 is a multifunctional protein involved in several steps of the progression of cell transformation, these data on structure and folding pathways are therefore crucial to designing anticancer drugs targeting AnxA2.

Annexin A2 binds the internal ribosomal entry site of c-myc mRNA and regulates its translation.

The expression and localization of the oncoprotein c-Myc is highly regulated at the level of transcription, mRNA transport, translation, as well as stability of the protein. We previously showed that Annexin A2 (AnxA2) binds to a specific localization element in the 3'untranslated region (UTR) of c-myc mRNA and is involved in its localization to the perinuclear region. In the present study, we demonstrate that AnxA2 binds in a Ca2+-dependent manner to the internal ribosomal entry site (IRES) containing two pseudo-knots in the 5´UTR of the c-myc mRNA. Here, we employ an in vitro rabbit reticulocyte lysate system with chimeric c-myc reporter mRNAs to demonstrate that binding of AnxA2 to the c-myc IRES modulates the expression of c-Myc. Notably, we show that low levels of AnxA2 appear to increase, while high levels of AnxA2 inhibits translation of the chimeric mRNA. However, when both the AnxA2-binding site and the ribosomal docking site in the c-myc IRES are deleted, AnxA2 has no effect on the translation of the reporter mRNA. Forskolin-treatment of PC12 cells results in upregulation of Ser25 phosphorylated AnxA2 expression while c-Myc expression is down-regulated. The effect of forskolin on c-Myc expression and the level of Ser25 phosphorylated AnxA2 was abolished in the presence of EGTA. These findings indicate that AnxA2 regulates both the transport and subsequent translation of the c-myc mRNA, possibly by silencing the mRNA during its transport. They also suggest that AnxA2 act as a switch to turn off the c-myc IRES activity in the presence of calcium.Abbreviations: AnxA2, Annexin A2; ß2--µglob, ß2-microglobulin; cpm, counts per minute; hnRNP, heterogenous nuclear ribonucleoprotein; IRES, internal ribosomal entry site; ITAF, IRES trans-acting factor; MM, multiple myeloma; PABP, poly(A)-binding protein; PCBP, poly(rC) binding protein; PSF, PTB-associated splicing factor; PTB, polypyrimidine tract binding protein; RRL, rabbit reticulocyte lysate; UTR, untranslated region; YB, Y-box binding protein.

Co-localization of Interleukin-1α and Annexin A2 at the plasma membrane in response to oxidative stress.

Interleukin-1α (IL-1α) and Annexin A2 (AnxA2) are pleiotropic molecules with both intracellular and extracellular roles. They share several characteristics including unconventional secretion aided by S100 proteins, anchoring of the externalized proteins at the outer surface of the plasma membrane and response to oxidative stress. Although IL-1α and AnxA2 have been implicated in a variety of biological processes, including cancer, little is known about the mechanisms of their cellular release. In the present study, employing the non-cancerous breast epithelial MCF10A cells, we demonstrate that IL-1α and AnxA2 establish a close association in response to oxidative stress. Stress conditions lead to translocation of both proteins towards lamellipodia rich in vimentin and association of full-length IL-1α and Tyr23 phosphorylated AnxA2 with the plasma membrane at peripheral sites depleted of F-actin. Notably, membrane-associated IL-1α and AnxA2 preferentially localize to the outer edges of the MCF10A cell islands, suggesting that the two proteins participate in the communication of these epithelial cells with their neighboring cells. Similarly, in U2OS osteosarcoma cell line both endogenous IL-1α and transiently produced IL-1α/EGFP associate with the plasma membrane. While benign MFC10A cells present membrane-associated IL-1α and AnxA2 at the edges of their cell islands, the aggressive cancerous U2OS cells communicate in such manner also with distant cells.

Reactive oxygen species exert opposite effects on Tyr23 phosphorylation of the nuclear and cortical pools of annexin A2.

Annexin A2 (AnxA2) is a multi-functional and -compartmental protein whose subcellular localisation and functions are tightly regulated by its post-translational modifications. AnxA2 and its Tyr23-phosphorylated form (pTyr23AnxA2) are involved in malignant cell transformation, metastasis and angiogenesis. Here, we show that H2O2 exerts rapid, simultaneous and opposite effects on the Tyr23 phosphorylation status of AnxA2 in two distinct compartments of rat pheochromocytoma (PC12) cells. Reactive oxygen species induce dephosphorylation of pTyr23AnxA2 located in the PML bodies of the nucleus, whereas AnxA2 associated with F-actin at the cell cortex is Tyr23 phosphorylated. The H2O2-induced responses in both compartments are transient and the pTyr23AnxA2 accumulating at the cell cortex is subsequently incorporated into vesicles and then released to the extracellular space. Blocking nuclear export by leptomycin B does not affect the nuclear pool of pTyr23AnxA2, but increases the amount of total AnxA2 in this compartment, indicating that the protein might have several functions in the nucleus. These results suggest that Tyr23 phosphorylation can regulate the function of AnxA2 at distinct subcellular sites.

Characterization of interactions between hepatitis C virus NS5B polymerase, annexin A2 and RNA - effects on NS5B catalysis and allosteric inhibition.

BACKGROUND: Direct acting antivirals (DAAs) provide efficient hepatitis C virus (HCV) therapy and clearance for a majority of patients, but are not available or effective for all patients. They risk developing HCV-induced hepatocellular carcinoma (HCC), for which the mechanism remains obscure and therapy is missing. Annexin A2 (AnxA2) has been reported to co-precipitate with the non-structural (NS) HCV proteins NS5B and NS3/NS4A, indicating a role in HCC tumorigenesis and effect on DAA therapy. METHODS: Surface plasmon resonance biosensor technology was used to characterize direct interactions between AnxA2 and HCV NS5B, NS3/NS4 and RNA, and the subsequent effects on catalysis and inhibition. RESULTS: No direct interaction between AnxA2 and NS3/NS4A was detected, while AnxA2 formed a slowly dissociating, high affinity (K D = 30 nM), complex with NS5B, decreasing its catalytic activity and affinity for the allosteric inhibitor filibuvir. The RNA binding of the two proteins was independent and AnxA2 and NS5B interacted with different RNAs in ternary complexes of AnxA2:NS5B:RNA, indicating specific preferences. CONCLUSIONS: The complex interplay revealed between NS5B, AnxA2, RNA and filibuvir, suggests that AnxA2 may have an important role for the progression and treatment of HCV infections and the development of HCC, which should be considered also when designing new allosteric inhibitors.

Protein phosphorylation and its role in the regulation of Annexin A2 function.

BACKGROUND: Annexin A2 (AnxA2) is a multifunctional protein involved in endocytosis, exocytosis, membrane domain organisation, actin remodelling, signal transduction, protein assembly, transcription and mRNA transport, as well as DNA replication and repair. SCOPE OF REVIEW: The current knowledge of the role of phosphorylation in the functional regulation of AnxA2 is reviewed. To provide a more comprehensive treatment of this topic, we also address in depth the phosphorylation process in general and discuss its possible conformational effects. Furthermore, we discuss the apparent limitations of the methods used to investigate phosphoproteins, as exemplified by the study of AnxA2. MAJOR CONCLUSIONS: AnxA2 is subjected to complex regulation by post-translational modifications affecting its cellular functions, with Ser11, Ser25 and Tyr23 representing important phosphorylation sites. Thus, Ser phosphorylation of AnxA2 is involved in the recruitment and docking of secretory granules, the regulation of its association with S100A10, and sequestration of perinuclear, translationally inactive mRNP complexes. By contrast, Tyr phosphorylation of AnxA2 regulates its role in actin dynamics and increases its association with endosomal compartments. Modification of its three main phosphorylation sites is not sufficient to discriminate between its numerous functions. Thus, fine-tuning of AnxA2 function is mediated by the joint action of several post-translational modifications. GENERAL SIGNIFICANCE: AnxA2 participates in malignant cell transformation, and its overexpression and/or phosphorylation is associated with cancer progression and metastasis. Thus, tight regulation of AnxA2 function is an integral aspect of cellular homeostasis. The presence of AnxA2 in cancer cell-derived exosomes, as well as the potential regulation of exosomal AnxA2 by phosphorylation or other PTMs, are topics of great interest.

Influenza A virus protein PB1-F2 from different strains shows distinct structural signatures.

The proapoptotic influenza A virus PB1-F2 protein contributes to viral pathogenicity and is present in most human and avian influenza isolates. The structures of full-length PB1-F2 of the influenza strains Pandemic flu 2009 H1N1, 1918 Spanish flu H1N1, Bird flu H5N1 and H1N1 PR8, have been characterized by NMR and CD spectroscopy. The study was conducted using chemically synthesized full-length PB1-F2 protein and fragments thereof. The amino acid residues 30-70 of PR8 PB1-F2 were found to be responsible for amyloid formation of the protein, which could be assigned to formation of ß-sheet structures, although α-helices were the only structural features detected under conditions that mimic a membranous environment. At membranous conditions, in which the proteins are found in their most structured state, significant differences become apparent between the PB1-F2 variants investigated. In contrast to Pandemic flu 2009 H1N1 and PR8 PB1-F2, which exhibit a continuous extensive C-terminal α-helix, both Spanish flu H1N1 and Bird flu H5N1 PB1-F2 contain a loop region with residues 66-71 that divides the C-terminus into two shorter helices. The observed structural differences are located to the C-terminal ends of the proteins to which most of the known functions of these proteins have been assigned. A C-terminal helix-loop-helix motif might be a structural signature for PB1-F2 of the highly pathogenic influenza viruses as observed for 1918 Spanish flu H1N1 and Bird flu H5N1 PB1-F2. This signature could indicate the pathological nature of viruses emerging in the future and thus aid in the recognition of these viruses.

Arrivals and departures at the plasma membrane: direct and indirect transport routes.

Studies carried out during the last 2 decades have dramatically increased our knowledge of the pathways and mechanisms of intracellular membrane traffic, most recently due to the developments in light microscopy and in vivo imaging of fluorescent fusion proteins. These studies have also revealed that certain molecules do not behave according to the classical transportation rules first documented in cell biology textbooks in the 1980s and 1990s. Initially, unconventional mechanisms of secretion that do not involve passage of cargo through the stacked Golgi cisternae were thought to confer on cells the ability to discard excess amounts of protein products. With time, however, more physiological mechanisms and roles have been proposed for an increasing number of secretory processes that bypass the Golgi apparatus.

The flavagline FL3 interferes with the association of Annexin A2 with the eIF4F initiation complex and transiently stimulates the translation of annexin A2 mRNA.

Introduction: Annexin A2 (AnxA2) plays a critical role in cell transformation, immune response, and resistance to cancer therapy. Besides functioning as a calcium- and lipidbinding protein, AnxA2 also acts as an mRNA-binding protein, for instance, by interacting with regulatory regions of specific cytoskeleton-associated mRNAs. Methods and Results: Nanomolar concentrations of FL3, an inhibitor of the translation factor eIF4A, transiently increases the expression of AnxA2 in PC12 cells and stimulates shortterm transcription/translation of anxA2 mRNA in the rabbit reticulocyte lysate. AnxA2 regulates the translation of its cognate mRNA by a feed-back mechanism, which can partly be relieved by FL3. Results obtained using the holdup chromatographic retention assay results suggest that AnxA2 interacts transiently with eIF4E (possibly eIF4G) and PABP in an RNA-independent manner while cap pulldown experiments indicate a more stable RNA-dependent interaction. Short-term (2 h) treatment of PC12 cells with FL3 increases the amount of eIF4A in cap pulldown complexes of total lysates, but not of the cytoskeletal fraction. AnxA2 is only present in cap analogue-purified initiation complexes from the cytoskeletal fraction and not total lysates confirming that AnxA2 binds to a specific subpopulation of mRNAs. Discussion: Thus, AnxA2 interacts with PABP1 and subunits of the initiation complex eIF4F, explaining its inhibitory effect on translation by preventing the formation of the full eIF4F complex. This interaction appears to be modulated by FL3. These novel findings shed light on the regulation of translation by AnxA2 and contribute to a better understanding of the mechanism of action of eIF4A inhibitors.

RNA-binding is an ancient trait of the Annexin family.

Introduction: The regulation of intracellular functions in mammalian cells involves close coordination of cellular processes. During recent years it has become evident that the sorting, trafficking and distribution of transport vesicles and mRNA granules/complexes are closely coordinated to ensure effective simultaneous handling of all components required for a specific function, thereby minimizing the use of cellular energy. Identification of proteins acting at the crossroads of such coordinated transport events will ultimately provide mechanistic details of the processes. Annexins are multifunctional proteins involved in a variety of cellular processes associated with Ca2+-regulation and lipid binding, linked to the operation of both the endocytic and exocytic pathways. Furthermore, certain Annexins have been implicated in the regulation of mRNA transport and translation. Since Annexin A2 binds specific mRNAs via its core structure and is also present in mRNP complexes, we speculated whether direct association with RNA could be a common property of the mammalian Annexin family sharing a highly similar core structure. Methods and results: Therefore, we performed spot blot and UV-crosslinking experiments to assess the mRNA binding abilities of the different Annexins, using annexin A2 and c-myc 3'UTRs as well as c-myc 5'UTR as baits. We supplemented the data with immunoblot detection of selected Annexins in mRNP complexes derived from the neuroendocrine rat PC12 cells. Furthermore, biolayer interferometry was used to determine the KD of selected Annexin-RNA interactions, which indicated distinct affinities. Amongst these Annexins, Annexin A13 and the core structures of Annexin A7, Annexin A11 bind c-myc 3'UTR with KDs in the nanomolar range. Of the selected Annexins, only Annexin A2 binds the c-myc 5'UTR indicating some selectivity. Discussion: The oldest members of the mammalian Annexin family share the ability to associate with RNA, suggesting that RNA-binding is an ancient trait of this protein family. Thus, the combined RNA- and lipid-binding properties of the Annexins make them attractive candidates to participate in coordinated long-distance transport of membrane vesicles and mRNAs regulated by Ca2+. The present screening results can thus pave the way for studies of the multifunctional Annexins in a novel cellular context.

Structure of the ALS Mutation Target Annexin A11 Reveals a Stabilising N-Terminal Segment.

The functions of the annexin family of proteins involve binding to Ca2+, lipid membranes, other proteins, and RNA, and the annexins share a common folded core structure at the C terminus. Annexin A11 (AnxA11) has a long N-terminal region, which is predicted to be disordered, binds RNA, and forms membraneless organelles involved in neuronal transport. Mutations in AnxA11 have been linked to amyotrophic lateral sclerosis (ALS). We studied the structure and stability of AnxA11 and identified a short stabilising segment in the N-terminal end of the folded core, which links domains I and IV. The crystal structure of the AnxA11 core highlights main-chain hydrogen bonding interactions formed through this bridging segment, which are likely conserved in most annexins. The structure was also used to study the currently known ALS mutations in AnxA11. Three of these mutations correspond to buried Arg residues highly conserved in the annexin family, indicating central roles in annexin folding. The structural data provide starting points for detailed structure-function studies of both full-length AnxA11 and the disease variants being identified in ALS.

Two tales of Annexin A2 knock-down: One of compensatory effects by antisense RNA and another of a highly active hairpin ribozyme.

Besides altering its own expression during cell transformation, Annexin A2 is upregulated during the progression of many cancer types and also plays key roles during viral infection and multiplication. Consequently, there has been great interest in Annexin A2 as a potential drug target. The successful design of efficient in vivo delivery systems constitutes an obstacle in full exploitation of antisense and RNA-cleaving technologies for the knock-down of specific targets. Efficiency is dependent on the method of delivery and accessibility of the target. Here, hairpin ribozymes and an antisense RNA against rat annexin A2 mRNA were tested for their efficiencies in a T7-driven coupled transcription/translation system. The most efficient ribozyme and antisense RNA were subsequently inserted into a retroviral vector under the control of a tRNA promoter, in a cassette inserted between retroviral Long Terminal Repeats for stable insertion into host DNA. The Phoenix package system based on defective retroviruses was used for virus-mediated gene transfer into PC12 cells. Cells infected with the ribozyme-containing particles died shortly after infection. However, the same ribozyme showed a very high catalytic effect in vitro in cell lysates, explained by its loose hinge helix 2 region. This principle can be transferred to other ribozymes, such as those designed to cleave the guide RNA in the CRISPR/Cas9 technology, as well as to target specific viral RNAs. Interestingly, efficient down-regulation of the expression of Annexin A2 by the antisense RNA resulted in up-regulation of Annexin A7 as a compensatory effect after several cell passages. Indeed, compensatory effects have previously been observed during gene knock-out, but not during knock-down of protein expression. This highlights the problems in interpreting the phenotypic effects of knocking down the expression of a protein. In addition, these data are highly relevant when considering the effects of the CRISPR/Cas9 approach.

Cytotoxic saponins and other natural products from flowering tops of Narthecium ossifragum L.

For more than four centuries, the intake of Narthecium ossifragum has been associated with poisoning in domesticated animals. Saponins occurring in flowering tops of the plant are considered to cause kidney damage in calves. At present, there are more than 30 papers on the saponins of N. ossifragum in the literature, although the structures of these compounds have hitherto not been determined. Here, we identify the saponins of N. ossifragum as sarsasapogenin, sarsasapogenin-3-O-ß-galactopyranoside, sarsasapogenin-3-O-(2'-O-ß-glucopyranosyl-ß-galactopyranoside) and sarsasapogenin-3-O-(2'-O-ß-glucopyranosyl-3'-O-α-arabinopyranosyl-ß-galactopyranoside). Moreover, six aromatic natural products were isolated and characterized from the methanolic extract from flowers of N. ossifragum. Five of these aromatic compounds, chrysoeriol 6-C-ß-arabinofuranoside-8-C-ß-glucopyranoside, chrysoeriol 6-C-ß-arabinopyranosyl-8-C-ß-glucopyranoside, chrysoeriol 6-C-ß-xylopyranosyl-8-C-ß-galactopyranoside, chrysoeriol 6-C-ß-galactopyranosyl-8-C-ß-glucopyranoside and chrysoeriol 6-C-ß-glucopyranosyl-8-C-ß-galactopyranoside are undescribed. All compounds were tested for cytotoxicity in mammalian cell lines derived from the heart, kidney, and haematological tissues. The saponins exhibited cytotoxicity in the micromolar range, with proportionally increasing cytotoxicity with increasing number of glycosyl substituents. The most potent compound was the main saponin sarsasapogenin-3-O-(2'-O-ß-glucopyranosyl-3'-O-α-arabinopyranosyl-ß-galactopyranoside), which produced cell death at concentrations below 3-4 µM in all three cell lines tested. This indicates that the saponins are the toxicants mainly responsible for kidney damage observed in cattle after ingestion of N. ossifragum. Our findings also pave the way for analysis of individual compounds isolated during the biopsies of intoxicated animals.

The mRNA-binding site of annexin A2 resides in helices C-D of its domain IV.

Annexin A2 (AnxA2) is a Ca(2+)-binding and phospholipid-binding protein involved in different intracellular processes including exocytosis, endocytosis and membrane-cytoskeleton movements. We have previously identified AnxA2 as an mRNA-binding protein present in cytoskeleton-bound polysomes, that binds to a specific approximately 100 nucleotide region in the 3'-untranslated region of c-myc and its cognate mRNA. In the present study, we show by UV cross-linking assays and surface plasmon resonance analyses that the mRNA-binding site of AnxA2 resides in its domain IV. Furthermore, the interaction of full-length AnxA2 with the 3'-untranslated region of anxA2 mRNA is Ca(2+)-dependent. By contrast, the interaction is Ca(2+)-independent for the isolated domain IV of AnxA2, suggesting that the mRNA-binding site is masked in Apo-AnxA2 and gains exposure through a Ca(2+)-induced conformational change of AnxA2 generating a favourable mRNA-binding site. The AnxA2-mRNA interaction is specific and involves helices C and D in domain IV of AnxA2, since point mutagenesis of several charged and polar exposed residues of these helices in the full-length protein strongly reduce RNA binding. The interaction appears to be sequential involving an initial phase of recognition dominated by electrostatic interactions, most likely between lysine residues and the phosphate backbone of RNA, followed by a second phase contributing to the specificity of the interaction.

Annexin A2 recognises a specific region in the 3'-UTR of its cognate messenger RNA.

Annexin A2 is a multifunctional Ca(2+)- and lipid-binding protein. We previously showed that a distinct pool of cellular Annexin A2 associates with mRNP complexes or polysomes associated with the cytoskeleton. Here we report in vitro and in vivo experiments showing that Annexin A2 present in this subset of mRNP complexes interacts with its cognate mRNA and c-myc mRNA, but not with beta(2)-microglobulin mRNA translated on membrane-bound polysomes. The protein recognises sequence elements within the untranslated regions, but not within the coding region, of its cognate mRNA. Alignment of the Annexin A2-binding 3'-untranslated regions of annexin A2 mRNA from several species reveals a five nucleotide consensus sequence 5'-AA(C/G)(A/U)G. The Annexin A2-interacting region of the 3'-untranslated region can be mapped to a sequence of about 100 nucleotides containing two repeats of the consensus sequence. The binding elements appear to involve both single and double stranded regions, indicating that a specific higher order mRNA structure is required for binding to Annexin A2. We suggest that this type of interaction is representative for a group of mRNAs translated on cytoskeleton-bound polysomes.

Engineering, biophysical characterisation and binding properties of a soluble mutant form of annexin A2 domain IV that adopts a partially folded conformation.

The four approximately 75-residue domains (repeats) that constitute the annexin core structure all possess an identical five-alpha-helix bundle topology, but the physico-chemical properties of the isolated domains are different. Domain IV of the annexins has previously been expressed only as inclusion bodies, resistant to solubilisation. Analysis of the conserved, exposed hydrophobic residues of the four annexin domains reveals that domain IV contains the largest number of hydrophobic residues involved in interfacial contacts with the other domains. We designed five constructs of domain IV of annexin A2 in which several interfacial hydrophobic residues were substituted by hydrophilic residues. The mutant domain, in which all fully exposed hydrophobic interfacial residues were substituted, was isolated as a soluble protein. Circular dichroism measurements indicate that it harbours a high content of alpha-helical secondary structure and some tertiary structure. The CD-monitored (lambda=222 nm) thermal melting profile suggests a weak cooperative transition. Nuclear magnetic resonance (1H-15N) correlation spectroscopy reveals heterogeneous line broadening and an intermediate spectral dispersion. These properties are indicative of a partially folded protein in which some residues are in a fairly structured conformation, whereas others are in an unfolded state. This conclusion is corroborated by 1-anilinonaphthalene-8-sulfonate fluorescence (ANS) analyses. Surface plasmon resonance measurements also indicate that this domain binds heparin, a known ligand of domain IV in the full-length annexin A2, although with lower affinity.

Post-translational modifications of Annexin A2 are linked to its association with perinuclear nonpolysomal mRNP complexes.

Various post-translational modifications (PTMs) regulate the localisation and function of the multifunctional protein Annexin A2 (AnxA2). In addition to its various tasks as a cytoskeletal- and membrane-associated protein, AnxA2 can function as a trans-acting protein binding to cis-acting sequences of specific mRNAs. In the present study, we have examined the role of Ser25 phosphorylation in subcellular localisation of AnxA2 and its interaction with mRNP complexes. Subcellular fractionation and confocal microscopy of rat neuroendocrine PC12 cells showed that Ser25-phosphorylated AnxA2 (pSer25AnxA2) is absent from the nucleus and mainly localised to the perinuclear region, evidently associating with both membranes and cytoskeletal elements. Perinuclear targeting of AnxA2 was abolished by inhibition of protein kinase C activity, which resulted in cortical enrichment of the protein. Although oligo(dT)-affinity purification of mRNAs revealed that pSer25AnxA2 associates with nonpolysomal, translationally inactive mRNP complexes, it displayed only partial overlap with a marker of P-bodies. The phosphorylated protein is present as high-molecular-mass forms, indicating that it contains additional covalent PTMs, apparently triggered by its Ser25 phosphorylation. The subcellular distributions of these forms clearly differ from the main form of AnxA2 and are also distinct from that of Tyr23-phosphorylated AnxA2. Immunoprecipitation verified that these high-molecular-mass forms are due to ubiquitination and/or sumoylation. Moreover, these results indicate that Ser25 phosphorylation and ubiquitin/SUMO1 conjugation of AnxA2 promote its association with nonpolysomal mRNAs, providing evidence of a possible mechanism to sequester a subpopulation of mRNAs in a translationally inactive and transport competent form at a distinct subcellular localisation.

Extracellular vesicles released from cells exposed to reactive oxygen species increase annexin A2 expression and survival of target cells exposed to the same conditions.

Annexin A2 (AnxA2) is present in multiple cellular compartments and interacts with numerous ligands including calcium, proteins, cholesterol, negatively charged phospholipids and RNA. These interactions are tightly regulated by its post-translational modifications. The levels of AnxA2 and its Tyr23 phosphorylated form (pTyr23AnxA2) are increased in many cancers and the protein is involved in malignant cell transformation, metastasis and angiogenesis. Our previous studies of rat pheochromocytoma (PC12) cells showed that reactive oxygen species (ROS) induce rapid, simultaneous and transient dephosphorylation of nuclear AnxA2, most likely associating with PML bodies, while AnxA2 associated with F-actin at the cell cortex undergoes Tyr23 phosphorylation. The pTyr23AnxA2 in the periphery of the cells is incorporated into intraluminal vesicles of multivesicular endosomes and subsequently released to the extracellular space. We show here that extracellular vesicles (EVs) from cells exposed to ROS prime untreated PC12 cells to better tolerate subsequent oxidative stress, thus enhancing their survival. There is an increase in the levels of pTyr23AnxA2 and AnxA2 in the primed cells, suggesting that AnxA2 is involved in their survival. This increase is due to an upregulation of AnxA2 expression both at the transcriptional and translational levels after relatively short term (2 h) exposure to primed EVs.