Shadoo binds lipid membranes and undergoes aggregation and fibrillization

Lipid membrane can enhance prion protein (PrP) pathological fibrillogenesis. A neuronal paralog of PrP, named Shadoo (Sho), is localized to similar membrane environment as PrP and can also convert to amyloid-like fibrilles. To gain insight into the role of Sho in prion diseases, we studied Sho interactions with cellular membrane models. Sho was found to bind anionic lipid vesicles. Spectroscopic and microscopic data showed that membrane-associated Sho slowly converted into amyloid fibers. Furthermore, binding of Sho to anionic liposomes has a disruptive effect on the integrity of the lipid bilayer leading to the formation of supramolecular lipid–protein complexes. In consequence, the role of Sho in prion diseases might depend on the oligomerization state of Sho but also the nature of these lipoprotein assembles.     

Annexin B12 is a sensor of membrane curvature and undergoes major curvature-dependent structural changes

The regulation of membrane curvature plays an important role in many membrane trafficking and fusion events. Recent studies have begun to identify some of the proteins involved in controlling and sensing the curvature of cellular membranes. A mechanistic understanding of these processes is limited, however, as structural information for the membrane-bound forms of these proteins is scarce. Here, we employed a combination of biochemical and biophysical approaches to study the interaction of annexin B12 with membranes of different curvatures. We observed selective and Ca(2+)-independent binding of annexin B12 to negatively charged vesicles that were either highly curved or that contained lipids with negative intrinsic curvature. This novel curvature-dependent membrane interaction induced major structural rearrangements in the protein and resulted in a backbone fold that was different from that of the well characterized Ca(2+)-dependent membrane-bound form of annexin B12. Following curvature-dependent membrane interaction, the protein retained a predominantly alpha-helical structure but EPR spectroscopy studies of nitroxide side chains placed at selected sites on annexin B12 showed that the protein underwent inside-out refolding that brought previously buried hydrophobic residues into contact with the membrane. These structural changes were reminiscent of those previously observed following Ca(2+)-independent interaction of annexins with membranes at mildly acidic pH, yet they occurred at neutral pH in the presence of curved membranes. The present data demonstrate that annexin B12 is a sensor of membrane curvature and that membrane curvature can trigger large scale conformational changes. We speculate that membrane curvature could be a physiological signal that induces the previously reported Ca(2+)-independent membrane interaction of annexins in vivo.     

Stu2p binds tubulin and undergoes an open-to-closed conformational change

Stu2p from budding yeast belongs to the conserved Dis1/XMAP215 family of microtubule-associated proteins (MAPs). The common feature of proteins in this family is the presence of HEAT repeat-containing TOG domains near the NH2 terminus. We have investigated the functions of the two TOG domains of Stu2p in vivo and in vitro. Our data suggest that Stu2p regulates microtubule dynamics through two separate activities. First, Stu2p binds to a single free tubulin heterodimer through its first TOG domain. A large conformational transition in homodimeric Stu2p from an open structure to a closed one accompanies the capture of a single free tubulin heterodimer. Second, Stu2p has the capacity to associate directly with microtubule ends, at least in part, through its second TOG domain. These two properties lead to the stabilization of microtubules in vivo, perhaps by the loading of tubulin dimers at microtubule ends. We suggest that this mechanism of microtubule regulation is a conserved feature of the Dis1/XMAP215 family of MAPs.     

The metastasis-promoting phosphatase PRL-3 shows activity toward phosphoinositides

Phosphatase of regenerating liver 3 (PRL-3) is suggested as a biomarker and therapeutic target in several cancers. It has a well-established causative role in cancer metastasis. However, little is known about its natural substrates, pathways, and biological functions, and only a few protein substrates have been suggested so far. To improve our understanding of the substrate specificity and molecular determinants of PRL-3 activity, the wild-type (WT) protein, two supposedly catalytically inactive mutants D72A and C104S, and the reported hyperactive mutant A111S were tested in vitro for substrate specificity and activity toward phosphopeptides and phosphoinositides (PIPs), their structural stability, and their ability to promote cell migration using stable HEK293 cell lines. We discovered that WT PRL-3 does not dephosphorylate the tested phosphopeptides in vitro. However, as shown by two complementary biochemical assays, PRL-3 is active toward the phosphoinositide PI(4,5)P(2). Our experimental results substantiated by molecular docking studies suggest that PRL-3 is a phosphatidylinositol 5-phosphatase. The C104S variant was shown to be not only catalytically inactive but also structurally destabilized and unable to promote cell migration, whereas WT PRL-3 promotes cell migration. The D72A mutant is structurally stable and does not dephosphorylate the unnatural substrate 3-O-methylfluorescein phosphate (OMFP). However, we observed residual in vitro activity of D72A against PI(4,5)P(2), and in accordance with this, it exhibits the same cellular phenotype as WT PRL-3. Our analysis of the A111S variant shows that the hyperactivity toward the unnatural OMFP substrate is not apparent in dephosphorylation assays with phosphoinositides: the mutant is completely inactive against PIPs. We observed significant structural destabilization of this variant. The cellular phenotype of this mutant equals that of the catalytically inactive C104S mutant. These results provide a possible explanation for the absence of the conserved Ser of the PTP catalytic motif in the PRL family. The correlation of the phosphatase activity toward PI(4,5)P(2) with the observed phenotypes for WT PRL-3 and the mutants suggests a link between the PI(4,5)P(2) dephosphorylation by PRL-3 and its role in cell migration.     

Myo1c binds phosphoinositides through a putative pleckstrin homology domain

Myo1c is a member of the myosin superfamily that binds phosphatidylinositol-4,5-bisphosphate (PIP(2)), links the actin cytoskeleton to cellular membranes and plays roles in mechano-signal transduction and membrane trafficking. We located and characterized two distinct membrane binding sites within the regulatory and tail domains of this myosin. By sequence, secondary structure, and ab initio computational analyses, we identified a phosphoinositide binding site in the tail to be a putative pleckstrin homology (PH) domain. Point mutations of residues known to be essential for polyphosphoinositide binding in previously characterized PH domains inhibit myo1c binding to PIP(2) in vitro, disrupt in vivo membrane binding, and disrupt cellular localization. The extended sequence of this binding site is conserved within other myosin-I isoforms, suggesting they contain this putative PH domain. We also characterized a previously identified membrane binding site within the IQ motifs in the regulatory domain. This region is not phosphoinositide specific, but it binds anionic phospholipids in a calcium-dependent manner. However, this site is not essential for in vivo membrane binding.     

The receptor binding domain of botulinum neurotoxin serotype C binds phosphoinositides

Botulinum neurotoxins (BoNTs) are the most toxic proteins known for humans and animals with an extremely low LD₅₀ of ∼1 ng/kg. BoNTs generally require a protein and a ganglioside on the cell membrane surface for binding, which is known as a “dual receptor” mechanism for host intoxication. Recent studies have suggested that in addition to gangliosides, other membrane lipids such as phosphoinositides may be involved in the interactions with the receptor binding domain (HCR) of BoNTs for better membrane penetration. Using two independent lipid-binding assays, we tested the interactions of BoNT/C-HCR with lipids in vitro domain. BoNT/C-HCR was found to bind negatively charged phospholipids, preferentially phosphoinositides in both assays. Interactions with phosphoinositides may facilitate tighter binding between neuronal membranes and BoNT/C.     

Lactadherin binds selectively to membranes containing phosphatidyl-L-serine and increased curvature

Lactadherin, a milk protein, contains discoidin-type lectin domains with homology to the phosphatidylserine-binding domains of blood coagulation factor VIII and factor V. We have found that lactadherin functions, in vitro, as a potent anticoagulant by competing with blood coagulation proteins for phospholipid binding sites [J. Shi and G.E. Gilbert, Lactadherin inhibits enzyme complexes of blood coagulation by competing for phospholipid binding sites, Blood 101 (2003) 2628-2636]. We wished to characterize the membrane-binding properties that correlate to the anticoagulant capacity. We labeled bovine lactadherin with fluorescein and evaluated binding to membranes of composition phosphatidylserine/phosphatidylethanolamine/phosphatidylcholine, 4:20:76 supported by 2 mum diameter glass microspheres. Lactadherin bound saturably with an apparent KD of 3.3+/-0.4 nM in a Ca++ -independent manner. The number of lactadherin binding sites increased proportionally to the phosphatidylserine content over a range 0-2% and less rapidly for higher phosphatidylserine content. Inclusion of phosphatidylethanolamine in phospholipid vesicles did not enhance the apparent affinity or number of lactadherin binding sites. The number of sites was at least 4-fold higher on small unilamellar vesicles than on large unilamellar vesicles, indicating that lactadherin binding is enhanced by membrane curvature. Lactadherin bound to membranes with synthetic dioleoyl phosphatidyl-L-serine but not dioleoyl phosphatidyl-D-serine indicating stereoselective recognition of phosphatidyl-L-serine. We conclude that lactadherin resembles factor VIII and V with stereoselective preference for phosphatidyl-L-serine and preference for highly curved membranes.     

Immobilized inhibitor-1 binds and inhibits protein phosphatase 1

Inhibitor-1 is a potent and specific inhibitor of protein phosphatase 1. Phosphorylation by cAMP-dependent protein kinase is required for expression of its inhibitor activity. In the present study, we have used immobilized inhibitor-1 preparations to study the mechanism underlying protein phosphatase 1 inhibition. Protein phosphatase 1 bound to phosphorylated inhibitor-1 covalently coupled to Sepharose or Affi-Gel beads but did not bind to immobilized preparations of dephosphorylated inhibitor-1 or bovine serum albumin. Phosphorylated inhibitor-1 coupled to Sepharose or Affi-Gel beads retained its ability to inhibit protein phosphatase 1, although the apparent IC50 was decreased about 500-fold. The extent of protein phosphatase 1 binding to immobilized phosphorylated inhibitor-1 was comparable to the degree of protein phosphatase inhibition when the inhibitor protein was present at a concentration near the IC50. The efficiency of protein phosphatase 1 binding to immobilized phosphorylated inhibitor-1 was dependent on the inhibitor concentration on the matrix. Taken together these data indicate that the inhibition of protein phosphatase 1 by phosphorylated inhibitor-1 is a consequence of the binding of the inhibitor protein to one or more sites on protein phosphatase 1.     

Cartilage aggrecan undergoes significant compositional and structural alterations during laryngeal cancer

Aggrecan is a key component of cartilage and is responsible for the integrity and function of the tissue. In this study, the content of aggrecan and its structural modifications in adjacent to cancer apparently normal cartilages (AANCs) from various stages of laryngeal squamous cell carcinoma (LSCC) were investigated. Our data demonstrated a stage-related loss of aggregable aggrecan in AANCs, compared to the healthy laryngeal cartilage (HLC), which was excessive in advanced stages of disease. On aggregable aggrecan level, AANCs were characterized by significant compositional and structural modifications, the extent of which was closely related with the stage of LSCC. Four concrete subpopulations of aggregable molecules with particular physicochemical characteristics were identified with a strong tendency to prevail subpopulations of molecules of lower hydrodynamic sizes with increasing LSCC stage. These findings demonstrated that the cleavage of aggregable aggrecan occurred in concrete peptide bonds within the CS-1 and CS-2 attachment domains. These significant alterations were closely associated with the process of cartilage destruction, indicating the crucial role of aggrecan during LSCC.     

The hepatic asialoglycoprotein receptor selectively binds to some endogenous tissues

The hepatic asialoglycoprotein receptor (ASGP-R) was isolated from various rat tissues or freshly prepared single cell suspensions and tested for the binding to endogenous tissues or specific cell types by indirect immunofluorescence. Inhibition with N-acetyl-D-galactosamine demonstrated specificity of binding. ASGP-R binds to mesodermal tissues and to selected cells of the majority of glandular tissues but not to lining epithelia. ASGP-R stains heart muscle but not skeletal muscle. In addition, ASGP-R stains spleen cells (52%), bone marrow cells (55%), thymocytes (62%), and a fraction of peripheral blood lymphocytes (29%), which was identified as B-lymphocytes. Five different rat tumors also showed binding of ASGP-R. The binding pattern and staining intensity of peanut agglutinin and soybean agglutinin were strikingly different although the binding specificity of these lectins is related to the ASPG-R. It is concluded that considerable numbers of endogenous binding sites for the hepatic ASGP-R exist in normal tissue, even on cells which pass the liver on circulation.     

Leupaxin binds to PEST domain tyrosine phosphatase PEP

PEST domain tyrosine phosphatase (PEP) is an intracellular protein tyrosine phosphatase and characterized by PEST motifs and proline-rich domains in the carboxyl terminal half. PEP is primarily expressed in hematopoietic cells, and together with PEP-binding Csk, may act as a negative regulator of antigen receptor signaling in lymphocytes. Here, we show the binding capability of PEP for leupaxin, which is preferentially expressed in hematopoietic cells and a comparatively new member of the paxillin family characterized by two protein-protein interaction modules, LIM domains and LD motifs. These results suggested that leupaxin might participate in the regulation of the signaling cascade through the binding to PEP in lymphocytes.     

Alpha-synuclein selectively binds to anionic phospholipids embedded in liquid-disordered domains

Previous studies indicate that binding of α-synuclein to membranes is critical for its physiological function and the development of Parkinson's disease (PD). Here, we have investigated the association of fluorescence-labeled α-synuclein variants with different types of giant unilamellar vesicles using confocal microscopy. We found that α-synuclein binds with high affinity to anionic phospholipids, when they are embedded in a liquid-disordered as opposed to a liquid-ordered environment. This indicates that not only electrostatic forces but also lipid packing and hydrophobic interactions are critical for the association of α-synuclein with membranes in vitro. When compared to wild-type α-synuclein, the disease-causing α-synuclein variant A30P bound less efficiently to anionic phospholipids, while the variant E46K showed enhanced binding. This suggests that the natural association of α-synuclein with membranes is altered in the inherited forms of Parkinson's disease.     

Porcine odorant-binding protein selectively binds to a human olfactory receptor

Odorant-binding proteins (OBPs) represent a highly abundant class of proteins secreted in the nasal mucus by the olfactory neuroepithelium. These proteins display binding affinity for a variety of odorant molecules, thereby assuming the role of carrier during olfactory perception. However, no specific interaction between OBP and olfactory receptors (ORs) has yet been shown and early events in olfaction remain so far poorly understood at a molecular level. Two human ORs, OR 17-209 and OR 17-210, were fused to a Green Fluorescent Protein and stably expressed in COS-7 cell lines. Interaction with OBP was investigated using a highly purified radioiodinated porcine OBP (pOBP) preparation, devoid of any ligand in its binding cavity. No specific binding of the pOBP tracer could be detected with OR 17-209. In contrast, OR 17-210 exhibited specific saturable binding (K(d) = 9.48 nM) corresponding to the presence of a single class of high-affinity binding sites (B(max) = 65.8 fmol/mg prot). Association and dissociation kinetics further confirmed high-affinity interaction between pOBP and OR 17-210. Autoradiographic studies of labeled pOBP to newborn mouse slices revealed the presence of multiple specific binding sites located mainly in olfactory tissue but also in several other peripheral tissues. Our data thus demonstrate a high-affinity interaction between OBP and OR, indicating that under physiological conditions, ORs may be specifically associated with an OBP partner in the absence of odorant. This provides further evidence of a novel role for OBP in the mechanism of olfactory perception.     

Insulin-like growth factor type I selectively binds to G-quadruplex structures

Background

G-quadruplex has been viewed as a promising therapeutic target in oncology due to its potentially important roles in physiological and pathological processes. Emerging evidence suggests that the biological functions of G-quadruplexes are closely related to the binding of some proteins. Insulin-like growth factor type I (IGF-1), as a significant modulator of cell growth and development, may serve as a quadruplex-binding protein.

Leupaxin binds to PEST domain tyrosine phosphatase PEP

PEST domain tyrosine phosphatase (PEP) is an intracellular protein tyrosine phosphatase and characterized by PEST motifs and proline-rich domains in the carboxyl terminal half. PEP is primarily expressed in hematopoietic cells, and together with PEP-binding Csk, may act as a negative regulator of antigen receptor signaling in lymphocytes. Here, we show the binding capability of PEP for leupaxin, which is preferentially expressed in hematopoietic cells and a comparatively new member of the paxillin family characterized by two protein-protein interaction modules, LIM domains and LD motifs. These results suggested that leupaxin might participate in the regulation of the signaling cascade through the binding to PEP in lymphocytes. (Mol Cell Biochem 269: 13–17, 2005)