Apo and Calcium-Bound Crystal Structures of Cytoskeletal Protein Alpha-14 Giardin (Annexin E1) from the Intestinal Protozoan Parasite Giardia lamblia

Alpha-14 giardin (annexin E1), a member of the alpha giardin family of annexins, has been shown to localize to the flagella of the intestinal protozoan parasite Giardia lamblia. Alpha giardins show a common ancestry with the annexins, a family of proteins most of which bind to phospholipids and cellular membranes in a Ca²⁺-dependent manner and are implicated in numerous membrane-related processes including cytoskeletal rearrangements and membrane organization. It has been proposed that alpha-14 giardin may play a significant role during the cytoskeletal rearrangement during differentiation of Giardia. To gain a better understanding of alpha-14 giardin's mode of action and its biological role, we have determined the three-dimensional structure of alpha-14 giardin and its phospholipid-binding properties. Here, we report the apo crystal structure of alpha-14 giardin determined in two different crystal forms as well as the Ca²⁺-bound crystal structure of alpha-14 giardin, refined to 1.9, 1.6 and 1.65 Å, respectively. Although the overall fold of alpha-14 giardin is similar to that of alpha-11 giardin, multiwavelength anomalous dispersion phasing was required to solve the alpha-14 giardin structure, indicating significant structural differences between these two members of the alpha giardin family. Unlike most annexin structures, which typically possess N-terminal domains, alpha-14 giardin is composed of only a core domain, followed by a C-terminal extension that may serve as a ligand for binding to cytoskeletal protein partners in Giardia. In the Ca²⁺-bound structure we detected five bound calcium ions, one of which is a novel, highly coordinated calcium-binding site not previously observed in annexin structures. This novel high-affinity calcium-binding site is composed of seven protein donor groups, a feature rarely observed in crystal structures. In addition, phospholipid-binding assays suggest that alpha-14 giardin exhibits calcium-dependent binding to phospholipids that coordinate cytoskeletal disassembly/assembly during differentiation of the parasite.     

Annexin 1 is secreted by the human prostate

The human prostate expresses very high concentrations of annexins 1, 4, and 5 and secretes high concentrations of annexins 1 and 5. Although the biological roles of these proteins in prostate secretions are not known, these studies emphasize the need to consider extracellular sites for physiological functions of annexins. The clear demonstration of secretion of proteins that have blocked N-termini and lack hydrophobic signal sequences raises the possibility that novel cellular secretory pathways exist. Preliminary immunohistochemical experiments in collaboration with Dr. James Fallon indicate that both annexins 1 and 4 are expressed in prostate ductal secretory epithelium. Since annexin 1, but not annexin 4, is secreted, a comparison of the cellular fate of these two related proteins in the prostate may provide a useful model system for determining the structural elements that direct the secretion of proteins which lack conventional signal sequences.     

Annexin A1 processing is associated with caspase-dependent apoptosis in BZR cells

Annexins are widely distributed and have been described in lung as well as in other cells and tissues. Annexin I (ANX AI) is a member of the calcium-dependent phospholipid binding protein family. Besides its anti-inflammatory function, ANX AI has been involved in several mechanisms such as the Erk repression pathway or apoptosis. To investigate the role of ANX AI on apoptosis in broncho-alveolar cells, we have constructed a plasmid containing the ANX AI full length cDNA. Transfected BZR cells displayed a higher level of both forms of ANX AI (37 and 33 kDa) as well as a decrease in cell viability (two-fold versus cells transfected with an empty vector). In order to analyse the endogenous ANX AI processing during stimulus-induced apoptosis, BZR cells were treated with a commonly used inducer, i.e. C2 ceramides. In these conditions, microscopic analysis revealed chromatin condensation in dying cells and the Bcl-2, Bcl-x(L)/Bax mRNA balance was altered. Caspase-3 is one of the key executioners of apoptosis, being responsible for the cleavage of many proteins such as the nuclear enzyme poly(ADP-ribose) polymerase (PARP). We demonstrate that caspase-3 was activated after 4 h treatment in the presence of ceramide leading to the cleavage of PARP. Dose-response experiments revealed that cell morphology and viability modifications following ceramide treatment were accompanied by an increase in endogenous ANX AI processing. Interestingly, in both ceramide and transfection experiments, the ANX AI cleaved form was enhanced whereas pre-treatment with the caspase inhibitor Z-VAD-fmk abolished ANX AI cleavage. In conclusion, this study demonstrates a complex regulatory role of caspase-dependent apoptosis where ANX AI is processed at the N-terminal region which could give susceptibility to apoptosis upon ceramide treatment.     

Alpha-1-antitrypsin-deficient phenotype is not maintained by segregation distortion

Recent reports have suggested that the alpha-1-antitrypsin allele PiZ, which in homozygotes results in severe deficiency of this important protease inhibitor, is maintained at a relatively high gene frequency through the mechanism of segregation distortion. We report here on 121 nuclear families selected because only one parent was segregating the Z allele. After correcting for ascertainment, no evidence of preferential transmission was observed in 278 informative offspring.     

Annexin I is an endogenous ligand that mediates apoptotic cell engulfment

Engulfment of apoptotic cells requires presentation of new cell surface ligands by the dying cells. Using a differential proteomics technology, we identify that annexin I is a caspase-dependent engulfment ligand; it is recruited from the cytosol and exported to the outer plasma membrane leaflet, colocalizes with phosphatidylserine, and is required for efficient clearance of apoptotic cells. Furthermore, phosphatidylserine receptor (PSR) clustering around apoptotic cells indicates a requirement for annexin I. In the nematode Caenorhabditis elegans, downregulation of the annexin homolog prevents efficient engulfment of pharyngeal cell corpses. These results provide novel mechanistic insights into how apoptotic cells are removed and may explain a pathogenic mechanism of chronic inflammatory diseases where annexin I autoantibodies have been described.     

Molecular Cloning and Characterization of Annexin V-Binding Proteins with Highly Hydrophilic Peptide Structure

Abstract: We previously reported that annexin V promoted the survival of cultured rat neocortical neurons. In an effort to elucidate the mechanism underlying this neurotrophic activity of annexin V, we have attempted to identify the target or binding proteins of annexin V in neuronal cells. Herein, we screened an embryonic day 17 rat brain cDNA library by western blot using glutathione S -transferase-annexin V fusion protein as a probe and then isolated four clones showing binding to annexin V in a Ca²⁺ - and phospholipid-dependent manner. Although these cDNAs encoded different polypeptides, all four polypeptides shared the unique feature of containing highly hydrophilic stretches with high Lys, Glu, and Ser contents. Deduced amino acid sequences of two clones showed high homology with human X-linked Helicase2 (XH2) and DNA (cytosine-5) methyltransferase (DMTase) sequences, whereas the other two were not related to any known peptide sequence. These results suggest that XH2 and DMTase are candidates for annexin V-binding proteins and thus may mediate the biological activity of annexin V.     

α1-抗胰蛋白酶的研究进展

自1963年首次报道α1-抗胰蛋白酶(α1-AT)缺失与肺气肿的关系以来,α1-AT的结构和功能、作用原理等得到进一步的认识。伴随α1-AT产品的不断研制,对其临床应用及其临床研究更加广泛深入,对此进行综述。     

Annexin A1 is regulated by domains cross-talk through post-translational phosphorylation and SUMOYlation

Mouse prostate membrane-associated proteins of the annexin family showed changes in SUMOylation during androgen treatment. Among these the calcium-binding annexin A1 protein (ANXA1) was chosen for further characterization given its role in protein secretion and cancer. SUMOylation of ANXA1 was confirmed by overexpressing SUMO-1 in LNCaP cells. Site-directed mutagenesis indicated that K257 located in a SUMOylation consensus motif in the C-terminal calcium-binding DA3 repeat domain is SUMOylated. Mutation of the N-terminal Y21 decreased markedly the SUMOylation signal while EGF stimulation increased ANXA1 SUMOylation. A structural analysis of ANXA1 revealed that K257 is located in a hot spot where Ca^2 + and SUMO-1 bind and where a nuclear export signal and a polyubiquitination site are also present. Also, Y21 is buried inside an α-helix structure in the Ca^2 +-free conformation implying that Ca^2 + binding, and the subsequent expelling of the N-terminal α-helix in a disordered conformation, is permissive for its phosphorylation. These results show for the first time that SUMOylation can be regulated by an external signal (EGF) and indicate the presence of a cross-talk between the N-terminal and C-terminal domains of ANXA1 through post-translational modifications.     

膜联蛋白A1及其与肿瘤关系的研究进展

膜联蛋白A1 (Annexin A1,ANXA1)是一种来源于脊柱(哺乳)动物的钙依赖性磷脂结合蛋白,是介导细胞内糖皮质激素抗炎作用的效应分子,在组织中广泛表达,参与细胞生长周期的各个阶段.其既可以可溶性形式存在,也可稳定或可逆结合于细胞骨架蛋白,调控细胞与细胞外基质的相互作用.大量的研究发现,AnnexinA1的表达在不同肿瘤组织中有差异,并且同一肿瘤不同类型中表达也不一样,其异常表达及细胞内定位改变可能跟多种恶性肿瘤的分化及转移相关.Annexin A1与肿瘤的密切关系,或许可使其发展为一个新的肿瘤标志物,为肿瘤的早期诊断、治疗及预后提供新的判断标准.因此,探讨Annexin A1与肿瘤的关系极具临床应用前景.     

Annexin A1 is a cell-intrinsic metalloregulator of zinc in human ILC2s

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Annexin A6 is an organizer of membrane microdomains to regulate receptor localization and signalling

Annexin A6 (AnxA6) belongs to the conserved annexin protein family--a group of Ca(2+) -dependent membrane binding proteins. It is the largest of all annexin proteins and upon activation, binds to negatively charged phospholipids in the plasma membrane and endosomes. In addition, AnxA6 associates with cholesterol-rich membrane microdomains termed lipid rafts. Membrane cholesterol triggers Ca(2+) -independent translocation of AnxA6 to membranes and AnxA6 levels determine the number of caveolae, a form of specialized rafts at the cell surface. AnxA6 also has an F-actin binding domain and interacts with cytoskeleton components. Taken together, this suggests that AnxA6 has a scaffold function to link membrane microdomains with the organization of the cytoskeleton. Such a link facilitates AnxA6 to participate in plasma membrane repair and it would also impact on receptor signalling at the cell surface, growth factor, and lipoprotein receptor trafficking, Ca(2+) -channel activity and T cell activation. Hence, the regulation of cell surface receptors by AnxA6 may be facilitated by its unique structure that allows recruitment of interaction partners and simultaneously bridging specialized membrane domains with cortical actin surrounding activated receptors.     

Annexin A7 is correlated with better clinical outcomes of patients with breast cancer

The clinical prognosis of breast cancer (BC) patients remains poor. Studies on BC microarrays GSE37751, GSE7390, and GSE21653 were reanalyzed to characterize the expressions of annexin A7 (ANXA7) in BC patients and the corresponding normal breast tissues and the correlation between ANXA7 expression and clinical characteristics and survivals of BC patients. Gene set enrichment analysis (GSEA) was applied to investigate the exact mechanisms as for the expression of ANXA7 and the proliferation of BC cells. The level of ANXA7 expression was significantly decreased in BC patients than that in normal controls (P < .0001). BC patients in the ANXA7 high‐expression group were associated with better clinical features such as tumor size; histopathological grading; estrogen receptors; and clinical risk groups according to St Gallen criteria, Nottingham prognostic index criteria, and Veridex signature compared with those in the ANXA7 low‐expression group. Higher expression of ANXA7 predicted better prognosis of BC patients. The result of GSEA indicated that ANXA7 might inhibit the proliferation of BC cells through biological processes involved in androgen response, heme metabolism, and oxidative phosphorylation. The messenger RNA and protein levels of ANXA7 were decreased in BC tissues compared with those in normal breast tissues. Our results proved that ANXA7 was downregulated in BC cells and that a higher expression of ANXA7 was associated with better prognosis of BC patients.     

Identification of natural compounds targeting Annexin A2 with an anti-cancer effect

Annexin A2, a multifunctional tumor associated protein, promotes nuclear factor-kappa B (NF-κB) activation by interacting with NF-κB p50 subunit and facilitating its nuclear translocation. Here we demonstrated that two ginsenosides Rg5 (G-Rg5) and Rk1 (G-Rk1), with similar structure, directly bound to Annexin A2 by molecular docking and cellular thermal shift assay. Both Rg5 and Rk1 inhibited the interaction between Annexin A2 and NF-κB p50 subunit, their translocation to nuclear and NF-κB activation. Inhibition of NF-κB by these two ginsenosides decreased the expression of inhibitor of apoptosis proteins (IAPs), leading to caspase activation and apoptosis. Over expression of K302A Annexin A2, a mutant version of Annexin A2, which fails to interact with G-Rg5 and G-Rk1, effectively reduced the NF-κB inhibitory effect and apoptosis induced by G-Rg5 and G-Rk1. In addition, the knockdown of Annexin A2 largely enhanced NF-κB activation and apoptosis induced by the two molecules, indicating that the effects of G-Rg5 and G-Rk1 on NF-κB were mainly mediated by Annexin A2. Taken together, this study for the first time demonstrated that G-Rg5 and G-Rk1 inhibit tumor cell growth by targeting Annexin A2 and NF-κB pathway, and G-Rg5 and G-Rk1 might be promising natural compounds for targeted cancer therapy.     

Alpha-1-proteinase inhibitor is a heparin binding serpin: molecular interactions with the Lys rich cluster of helix-F domain

Alpha-1-proteinase (alpha-1-PI) inhibitor is the major circulating serine protease inhibitor in humans. The porcine elastase and trypsin inhibitory activity of human and ovine alpha-1-PI is activated several fold in the presence of anti-coagulant heparin. The activation is allosteric and appears to be characterized by two steps of binding; a weak followed by a strong binding. The Kass for ovine and human alpha-1-PI inhibition of porcine pancreatic elastase was increased approximately 45 fold and 38 fold respectively. Using a combinatorial approach of multiple sequence alignment, surface topology, chemical modification and tryptic peptide mapping to identify the sequence of the heparin bound peptide; we demonstrate that heparin binds to the lysyl rich region of the F-helix of alpha-1-PI, which differs from that of heparin-antithrombin (AT) interactions. Molecular docking prediction using the MEDock algorithm approximates the three positively charged lysines (K154, K155, K174) of human alpha-1-PI in this interaction. This heparin alpha-1-PI interaction has been exploited to develop an affinity purification method, which can be used universally to obtain homogenous preparations of mammalian alpha-1-PIs useful for augmentation therapy. Collectively, all these findings imply that alpha-1-PI has a major role in regulating extra cellular protease activity and the physiological activator is heparin.     

Annexin B1 from Taenia solium metacestodes is a newly characterized member of the annexin family

We previously reported cloning of the Taenia solium annexin B1 gene from a metacestode cDNA expression library and demonstrated that it acts as a protective antigen for effective vaccine development against cysticercosis. In the present study we produced recombinant annexin B1 and antiserum against the protein to investigate its structural and functional properties. Western blotting of metacestode fractions indicated that T. solium annexin B1, similar to vertebrate annexins, associates with acid phospholipids in the presence of Ca(2+). This property was confirmed by the recognition of apoptotic cells by labeled annexin B1. CD spectroscopy results demonstrated that alpha-helices are the main secondary structures of the protein. Ca(2+) binding increases the alpha-helix content and causes significant thermal stabilization with a melting temperature increase of approximately 10 degrees C. Functional Ca(2+)-dependent phospholipid binding sites of annexin B1 were investigated using mutant proteins. By changing a conserved acidic amino acid residue that putatively combines Ca(2+) in each domain of annexin B1 singly or in combination, we found that Ca(2+) binding in the first domain is more important than that at the other Ca(2+) binding sites. Annexin B1 is a metacestode stage-specific antigen, with the protein being mainly localized in the teguments and surrounding cyst wall of T. solium metacestodes, suggesting a role in the parasite-host interaction.     

Annexin VI: an intracellular target for ATP

Annexin VI (AnxVI), an Ca2+- and phospholipid-binding protein, interacts in vitro with ATP in a calcium-dependent manner. Experimental evidence indicates that its nucleotide-binding domain which is localized in the C-terminal half of the protein differs structurally from ATP/GTP-binding motifs found in other nucleotide-binding proteins. The amino-acid residues of AnxVI directly involved in ATP binding have not been yet defined. Binding of ATP to AnxVI induces changes in the secondary and tertiary structures of protein, affecting the affinity of AnxVI for Ca2+ and, in consequence, influencing the Ca2+-dependent activities of AnxVI: binding to F-actin and to membranous phospholipids, and self-association of the annexin molecules. These observations suggest that ATP is a functional ligand for AnxVI in vivo, and ATP-sensitive AnxVI may play the role of a factor coupling vesicular transport and calcium homeostasis to cellular metabolism.     

Annexin II is a major component of fusogenic endosomal vesicles

We have used an in vitro assay to follow the proteins transferred from a donor to an acceptor upon fusion of early endosomes. The acceptor was a purified early endosomal fraction immunoisolated on beads and the donor was a metabolically-labeled early endosomal fraction in suspension. In the assay, both fractions were mixed in the presence of unlabeled cytosol, and then the beads were retrieved and washed. The donor proteins transferred to the acceptor were identified by two- dimensional gel electrophoresis and autoradiography. Approximately 50 major proteins were transferred and this transfer fulfilled all criteria established for endosome fusion in vitro. However, only a small subset of proteins was efficiently transferred, if donor endosomes were briefly sonicated to generate small (0.1 micron diam) vesicles before the assay. These include two acidic membrane proteins, and three alkaline peripheral proteins exposed on the cytoplasmic face of the membrane. Partial sequencing and Western blotting indicated that one of the latter components is annexin II, a protein known to mediate membrane-membrane interactions. Immunogold labeling of cryosections confirmed that annexin II is present on early endosomes in vivo. These data demonstrate that annexin II, together with the other four proteins we have identified, is a major component of fusogenic endosomal vesicles, suggesting that these proteins are involved in the binding and/or fusion process.