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.

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.

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.

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.

Ubiquitinated annexin A2 is enriched in the cytoskeleton fraction.

Annexin A2 is a multifunctional protein and its cellular functions are regulated by post-translational modifications and ligand binding. When purified from porcine intestinal mucosa and transformed mouse Krebs II cells, SDS-PAGE revealed high-molecular-mass forms in addition to the 36 kDa protomer. These forms were identified as poly-/multi-ubiquitin conjugates of annexin A2, and ubiquitination represents a novel post-translational modification of this protein. Subcellular fractionation of mouse Krebs II cells revealed an enrichment of annexin A2-ubiquitin conjugates in the Triton X-100 resistant cytoskeleton fraction, suggesting that ubiquitinated annexin A2 may have a role associated with its function as an actin-binding protein.

Annexin A2 binds to the localization signal in the 3' untranslated region of c-myc mRNA.

Messenger RNA trafficking, which provides a mechanism for local protein synthesis, is dependent on cis-acting sequences in the 3' untranslated regions (3'UTRs) of the mRNAs concerned acting together with trans-acting proteins. The C-MYC transcription factor is a proto-oncogene product involved in cell proliferation, differentiation and apoptosis. Localization of c-myc mRNA to the perinuclear cytoplasm and its association with the cytoskeleton is determined by a signal in the 3'UTR. Here we show the specific binding of a trans-acting factor to the perinuclear localization element in the 3'UTR of c-myc mRNA and identify this protein as annexin A2. Gel retardation and UV cross-linking experiments showed that proteins in fibroblast extracts formed complexes with the region of c-myc 3'UTR implicated in localization; a protein of approximately 36 kDa exhibited specific, Ca(2+)-dependent binding. Binding was reduced by introduction of a mutation that abrogates localization. Using RNA-affinity columns followed by gel electrophoresis and mass spectrometry this protein was identified as annexin A2. The RNA-protein complex formed by cell extracts was further retarded by anti-(annexin A2). Purified annexin A2 bound to the same region of the c-myc 3'UTR but binding was reduced by introduction of a mutation, as with cell extracts. It is proposed that binding of annexin A2 to the localization signal in the c-myc mRNA leads to association with the cytoskeleton and perinuclear localization. The data indicate a novel functional role for the RNA-binding properties of annexin A2 in perinuclear localization of mRNA and the association with the cytoskeleton.

The native structure of annexin A2 peptides in hydrophilic environment determines their anti-angiogenic effects.

The progression of aggressive cancer occurs via angiogenesis and metastasis makes these processes important targets for the development of anti-cancer agents. However, recent studies have raised the concern that selective inhibition of angiogenesis results in a switch towards increased tumour growth and metastasis. Since Annexin A2 (AnxA2) is involved in both angiogenesis and metastasis, it may serve as an ideal target for the simultaneous inhibition of both processes. Based on the discovery that domains I (D(I)) and IV (D(IV)) of AnxA2 are potent inhibitors of angiogenesis, we designed seven peptides derived from these domains based on AnxA2 crystal structures. The peptides were expressed as fusion peptides to increase their folding and solubility. Light scattering, far-UV circular dichroism and thermal transition analyses were employed to investigate their aggregation tendencies, α-helical propensity and stability, respectively. 2,2,2-trifluoroethanol (50%) increased the α-helical propensities of all peptides, indicating that they may favour a hydrophobic environment, but did not enhance their thermal stability. D(I)-P2 appears to be the most stable and folded peptide in a hydrophilic environment. The secondary structure of D(I)-P2 was confirmed by nuclear magnetic resonance spectra. The effect of the seven AnxA2 peptides on the formation and integrity of capillary-like networks was studied in a co-culture system mimicking many of the angiogenesis-related processes. Notably, D(I)-P2 inhibited significantly network formation in this system, indicating that the folded D(I)-P2 peptide interferes with vascular endothelial growth factor-dependent pro-angiogenic processes. Thus, this peptide has the potential of being developed further as an anti-angiogenic drug.

Effect of serine phosphorylation and Ser25 phospho-mimicking mutations on nuclear localisation and ligand interactions of annexin A2.

Annexin A2 (AnxA2) interacts with numerous ligands, including calcium, lipids, mRNAs and intracellular and extracellular proteins. Different post-translational modifications participate in the discrimination of the functions of AnxA2 by modulating its ligand interactions. Here, phospho-mimicking mutants (AnxA2-S25E and AnxA2-S25D) were employed to investigate the effects of Ser25 phosphorylation on the structure and function of AnxA2 by using AnxA2-S25A as a control. The overall α-helical structure of AnxA2 is not affected by the mutations, since the thermal stabilities and aggregation tendencies of the mutants differ only slightly from the wild-type (wt) protein. Unlike wt AnxA2, all mutants bind the anxA2 3' untranslated region and ß-γ-G-actin with high affinity in a Ca(2+)-independent manner. AnxA2-S25E is not targeted to the nucleus in transfected PC12 cells. In vitro phosphorylation of AnxA2 by protein kinase C increases its affinity to mRNA and inhibits its nuclear localisation, in accordance with the data obtained with the phospho-mimicking mutants. Ca(2+)-dependent binding of wt AnxA2 to phosphatidylinositol, phosphatidylinositol-3-phosphate, phosphatidylinositol-4-phosphate and phosphatidylinositol-5-phosphate, as well as weaker but still Ca(2+)-dependent binding to phosphatidylserine and phosphatidylinositol-3,5-bisphosphate, was demonstrated by a protein-lipid overlay assay, whereas binding of AnxA2 to these lipids, as well as its binding to liposomes, is inhibited by the Ser25 mutations. Thus, introduction of a modification (mutation or phosphorylation) at Ser25 appears to induce a conformational change leading to increased accessibility of the mRNA- and G-actin-binding sites in domain IV independent of Ca(2+) levels, while the Ca(2+)-dependent binding of AnxA2 to phospholipids is attenuated.

Domains I and IV of annexin A2 affect the formation and integrity of in vitro capillary-like networks.

Annexin A2 (AnxA2) is a widely expressed multifunctional protein found in different cellular compartments. In spite of lacking a hydrophobic signal peptide, AnxA2 is found at the cell surface of endothelial cells, indicative of a role in angiogenesis. Increased extracellular levels of AnxA2 in tumours correlate with neoangiogenesis, metastasis and poor prognosis. We hypothesised that extracellular AnxA2 may contribute to angiogenesis by affecting endothelial cell-cell interactions and motility. To address this question, we studied the effect of heterotetrameric and monomeric forms of AnxA2, as well as its two soluble domains on the formation and maintenance of capillary-like structures by using an in vitro co-culture system consisting of endothelial and smooth muscle cells. In particular, addition of purified domains I and IV of AnxA2 potently inhibited the vascular endothelial growth factor (VEGF)-dependent formation of the capillary-like networks in a dose-dependent manner. In addition, these AnxA2 domains disrupted endothelial cell-cell contacts in preformed capillary-like networks, resulting in the internalisation of vascular endothelial (VE)-cadherin and the formation of VE-cadherin-containing filopodia-like structures between the endothelial cells, suggesting increased cell motility. Addition of monoclonal AnxA2 antibodies, in particular against Tyr23 phosphorylated AnxA2, also strongly inhibited network formation in the co-culture system. These results suggest that extracellular AnxA2, most likely in its Tyr phosphorylated form, plays a pivotal role in angiogenesis. The exogenously added AnxA2 domains most likely mediate their effects by competing with endogenous AnxA2 for extracellular factors necessary for the initiation and maintenance of angiogenesis, such as those involved in the formation/integrity of cell-cell contacts.

Multiple roles of annexin A2 in post-transcriptional regulation of gene expression.

Increasing evidence points to the participation of the multifunctional protein Annexin A2 (AnxA2) in mRNA localisation as well as the translation of certain mRNAs on cytoskeleton-bound polysomes, and thereby in the regulation of the biosynthesis of specific proteins, such as c-Myc and AnxA2 itself, which are linked to cellular transformation. AnxA2 is most likely activated by signalling pathways, which result in its post-translational modifications and modulate its binding to various ligands, including specific mRNAs. Positive and polar residues in helices C-D in domain IV of AnxA2 bind to cis-acting elements in the 3'-UTRs of its cognate, c-myc, collagen prolyl 4-hydroxylase-α(I) and N-methyl-D-aspartate R1 mRNAs, thus contributing to post-transcriptional regulation of the expression of specific genes. The cis-acting elements appear to constitute a higher order structure, frequently containing the consensus sequence 5'-AA(C/G)(A/U)G; however, non-canonical AnxA2 binding sites may also be involved. In the case of c-myc mRNA, the association with AnxA2 appears to regulate its localisation and translation. In addition, the binding of AnxA2 to a pseudoknot structure present in infectious bronchitis viral RNA results in reduced efficiency of -1 ribosomal frameshifting, indicating its recruitment as a host protein during viral infection. Finally, the association of AnxA2 with endosomes and exosomes suggests a role in co-ordinated transport of mRNA and vesicles, i.e. processes that respond to extracellular signals and are expected to employ multifunctional proteins.