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.     

Dual inhibition of EGFR at protein and activity level via combinatorial blocking of PI4KIIα as anti-tumor strategy

Our previous studies indicate that phosphatidylinositol 4-kinase IIα can promote the growth of multi-malignant tumors viaHER-2/PI3K andMAPK pathways.However, the molecular mechanisms of this pathway and its potential for clinical application remain unknown. In this study, we found that PI4KIIα could be an ideal combinatorial target for EGFR treatment via regulating EGFR degradation. Results showed that PI4KIIα knockdown reduced EGFR protein level, and the expression ofPI4KIIα shows a strong correlation with EGFR in human breast cancer tissues (r = 0.77, P<0.01). PI4KIIα knockdown greatly prolonged the effects and decreased the effective dosage ofAG-1478, a specific inhibitor of EGFR. In addition, it significantly enhanced AG1478-induced inhibition of tumor cell survival and strengthened the effect of the EGFR-targeting anti-cancer drug Iressa in xenograft tumor models. Mechanistically, we found that PI4KIIα suppression increased EGFR ligand-independent degradation. Quantitative proteomic analysis by stable isotope labeling with amino acids in cell culture (SILAC) and LC-MS/MS suggested that HSP90mediated the effect of PI4KIIα onEGFR. Furthermore, we found that combined inhibition of PI4KIIα and EGFR suppressed both PI3K/AKT and MAPK/ERK pathways, and resulted in downregulation of multiple oncogenes like PRDX2, FASN, MTA2, ultimately leading to suppression of tumor growth. Therefore,we conclude that combined inhibition of PI4KIIα and EGFR exerts a multiple anti-tumor effect. Dual inhibition of EGFR at protein and activity level via combinatorial blocking of PI4KIIα presents anovel strategy tocombatEGFR-dependent tumors.     

Assembly and Molecular Architecture of the Phosphoinositide 3-Kinase p85α Homodimer

Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that are activated by growth factor and G-protein-coupled receptors and propagate intracellular signals for growth, survival, proliferation, and metabolism. p85α, a modular protein consisting of five domains, binds and inhibits the enzymatic activity of class IA PI3K catalytic subunits. Here, we describe the structural states of the p85α dimer, based on data from in vivo and in vitro solution characterization. Our in vitro assembly and structural analyses have been enabled by the creation of cysteine-free p85α that is functionally equivalent to native p85α. Analytical ultracentrifugation studies showed that p85α undergoes rapidly reversible monomer-dimer assembly that is highly exothermic in nature. In addition to the documented SH3-PR1 dimerization interaction, we identified a second intermolecular interaction mediated by cSH2 domains at the C-terminal end of the polypeptide. We have demonstrated in vivo concentration-dependent dimerization of p85α using fluorescence fluctuation spectroscopy. Finally, we have defined solution conditions under which the protein is predominantly monomeric or dimeric, providing the basis for small angle x-ray scattering and chemical cross-linking structural analysis of the discrete dimer. These experimental data have been used for the integrative structure determination of the p85α dimer. Our study provides new insight into the structure and assembly of the p85α homodimer and suggests that this protein is a highly dynamic molecule whose conformational flexibility allows it to transiently associate with multiple binding proteins.     

Structural Basis of DEPTOR to Recognize Phosphatidic Acid Using its Tandem DEP Domains

DEP domain containing mTOR-interacting protein (DEPTOR) plays pivotal roles in regulating metabolism, growth, autophagy and apoptosis by functions as an endogenous inhibitor of mTOR signaling pathway. Activated by phosphatidic acid, a second messenger in mTOR signaling, DEPTOR dissociates from mTORC1 complex with unknown mechanism. Here, we present a 1.5 Å resolution crystal structure, which shows that the N-terminal two tandem DEP domains of hDEPTOR fold into a dumbbell-shaped structure, protruding the characteristic β-hairpin arms of DEP domains on each side. An 18 amino acids DDEX motif at the end of DEP2 interacts with DEP1 and stabilizes the structure. Biochemical studies showed that the tandem DEP domains directly interact with phosphatidic acid using two distinct positively charged patches. These results provide insights into mTOR activation upon phosphatidic acid stimulation.     

Lysoglycerophospholipids in chronic inflammatory disorders: The PLA2/LPC and ATX/LPA axes

Lysophosphatidylcholine (LPC) and lysophosphatidic acid (LPA), the most prominent lysoglycerophospholipids, are emerging as a novel class of inflammatory lipids, joining thromboxanes, leukotrienes and prostaglandins with which they share metabolic pathways and regulatory mechanisms. Enzymes that participate in LPC and LPA metabolism, such as the phospholipase A₂ superfamily (PLA₂) and autotaxin (ATX, ENPP2), play central roles in regulating LPC and LPA levels and consequently their actions. LPC/LPA biosynthetic pathways will be briefly presented and LPC/LPA signaling properties and their possible functions in the regulation of the immune system and chronic inflammation will be reviewed. Furthermore, implications of exacerbated LPC and/or LPA signaling in the context of chronic inflammatory diseases, namely rheumatoid arthritis, multiple sclerosis, pulmonary fibrosis and hepatitis, will be discussed. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.     

The phosphoinositide sensitivity of the KV channel family

Recently, we screened several K_V channels for possible dependence on plasma membrane phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂). The channels were expressed in tsA-201 cells and the PI(4,5)P₂ was depleted by several manipulations in whole-cell experiments with parallel measurements of channel activity. In contrast to reports on excised-patches using Xenopus laevis oocytes, we found only K_V7, but none of the other tested K_V channels, to be strongly dependent on PI(4,5)P₂. We now have extended our study to K_V1.2 channels, a K_V channel we had not previously tested, because a new published study on excised patches showed regulation of the voltage-dependence of activation by PI(4,5)P₂. In full agreement with those published results, we found a reduction of current amplitude by ~20% after depletion of PI(4,5)P₂ and a small left shift in the activation curve of K_V1.2 channels. We also found a small reduction of K_V11.1 (hERG) currents that was not accompanied by a gating shift. In conclusion, our whole-cell methods yield a PI(4,5)P₂-dependence of K_V1.2 currents in tsA-201 cells that is comparable to findings from excised patches of Xenopus laevis oocytes. We discuss possible physiological rationales for PI(4,5)P₂ sensitivity of some ion channels and insensitivity of others.     

The superlattice model of lateral organization of membranes and its implications on membrane lipid homeostasis

Most biological membranes are extremely complex structures consisting of hundreds of different lipid and protein molecules. According to the famous fluid-mosaic model lipids and many proteins are free to diffuse very rapidly in the plane of the membrane. While such fast diffusion implies that different membrane lipids would be laterally randomly distributed, accumulating evidence indicates that in model and natural membranes the lipid components tend to adopt regular (superlattice-like) distributions. The superlattice model, put forward based on such evidence, is intriguing because it predicts that 1) there is a limited number of allowed compositions representing local minima in membrane free energy and 2) those energy minima could provide set-points for enzymes regulating membrane lipid compositions. Furthermore, the existence of a discrete number of allowed compositions could help to maintain organelle identity in the face of rapid inter-organelle membrane traffic.     

Binding of amphipathic α-helical antimicrobial peptides to lipid membranes: Lessons from temporins B and L

Temporins constitute a family of amphipathic α-helical antimicrobial peptides (AMP) and contain some of the shortest cytotoxic peptides, comprised of only 10-14 residues. General characteristics of temporins parallel those of other AMP, both in terms of structural features and biophysical properties relating to their interactions with membrane lipids, with selective lipid-binding properties believed to underlie the discrimination between target vs host cells. Lipid-binding properties also contribute to the cytotoxicity AMP, causing permeabilization of their target cell membranes. The latter functional property of AMP involves highly interdependent acidic phospholipid-induced conformational changes, aggregation, and formation of toxic oligomers in the membrane. These oligomers are subsequently converted to amyloid-type fibers, as demonstrated for e.g. temporins B and L in our laboratory, and more recently for dermaseptins by Auvynet et al. Amyloid state represents the generic minimum in the folding/aggregation free energy landscape, and for AMP its formation most likely serves to detoxify the peptides, in keeping with the current consensus on mature amyloid being inert and non-toxic. The above scenario is supported by sequence analyses of temporins as well as other amphipathic α-helical AMP belonging to diverse families. Accordingly, sequence comparison identifies ‘conformational switches’, domains with equal probabilities for adopting random coil, α-helical and β-sheet structures. These regions were further predicted also to aggregate and assemble into amyloid β-sheets. Taken together, the lipid-binding properties and structural characterization lend support to the notion that the mechanism of membrane permeabilization by temporins B and L and perhaps of most AMP could be very similar, if not identical, to that of the paradigm amyloid forming cytotoxic peptides, responsible for degenerative cell loss in e.g. prion, Alzheimer's and Parkinson's disease, and type 2 diabetes.     

Layilin, a cell surface hyaluronan receptor, interacts with merlin and radixin

Layilin is a widely expressed integral membrane hyaluronan receptor, originally identified as a binding partner of talin located in membrane ruffles. We have identified merlin, the neurofibromatosis type 2 tumor suppressor protein and radixin, as other interactors with the carboxy-terminal domain of layilin. We show that the carboxy-terminal domain of layilin is capable of binding to the amino-terminal domain of radixin. An interdomain interaction between the amino- and the carboxy-terminal domains of radixin inhibits its ability to bind to layilin. In the presence of acidic phospholipids, the interdomain interaction of radixin is inhibited and layilin can bind to full-length radixin. In contrast, layilin binds both full-length and amino-terminal merlin-GST fusion proteins without a requirement for phospholipids. Furthermore, layilin antibody can immunoprecipitate merlin, confirming association in vivo between these two proteins, which also display similar subcellular localizations in ruffling membranes. No interaction was observed between layilin and ezrin or layilin and moesin. These findings expand the known binding partners of layilin to include other members of the talin/band 4.1/ERM (ezrin, radixin, and moesin) family of cytoskeletal-membrane linker molecules. This in turn suggests that layilin may mediate signals from extracellular matrix to the cell cytoskeleton via interaction with different intracellular binding partners and thereby be involved in the modulation of cortical structures in the cell.