Isolated Variable Domains of an Antibody Can Assemble on Blood Coagulation Factor VIII into a Functional Fv-like Complex.

Single-chain variable fragments (scFv) are antigen-recognizing variable fragments of antibodies (FV) where both subunits (VL and VH) are connected via an artificial linker. One particular scFv, iKM33, directed against blood coagulation factor VIII (FVIII) was shown to inhibit major FVIII functions and is useful in FVIII research. We aimed to investigate the properties of iKM33 enabled with protease-dependent disintegration. Three variants of iKM33 bearing thrombin cleavage sites within the linker were expressed using a baculovirus system and purified by two-step chromatography. All proteins retained strong binding to FVIII by surface plasmon resonance, and upon thrombin cleavage, dissociated into VL and VH as shown by size-exclusion chromatography. However, in FVIII activity and low-density lipoprotein receptor-related protein 1 binding assays, the thrombin-cleaved iKM33 variants were still inhibitory. In a pull-down assay using an FVIII-affinity sorbent, the isolated VH, a mixture of VL and VH, and intact iKM33 were carried over via FVIII analyzed by electrophoresis. We concluded that the isolated VL and VH assembled into scFv-like heterodimer on FVIII, and the isolated VH alone also bound FVIII. We discuss the potential use of both protease-cleavable scFvs and isolated Fv subunits retaining high affinity to the antigens in various practical applications such as therapeutics, diagnostics, and research.

Characterization of interaction between blood coagulation factor VIII and LRP1 suggests dynamic binding by alternating complex contacts.

BACKGROUND: Deficiency in blood coagulation factor VIII (FVIII) results in life-threating bleeding (hemophilia A) treated by infusions of FVIII concentrates. To improve disease treatment, FVIII has been modified to increase its plasma half-life, which requires understanding mechanisms of FVIII catabolism. An important catabolic actor is hepatic low density lipoprotein receptor-related protein 1 (LRP1), which also regulates many other clinically significant processes. Previous studies showed complexity of FVIII site for binding LRP1. OBJECTIVES: To characterize binding sites between FVIII and LRP1 and suggest a model of the interaction. METHODS: A series of recombinant ligand-binding complement-type repeat (CR) fragments of LRP1 including mutated variants was generated in a baculovirus system and tested for FVIII interaction using surface plasmon resonance, tissue culture model, hydrogen-deuterium exchange mass spectrometry, and in silico. RESULTS: Multiple CR doublets within LRP1 clusters II and IV were identified as alternative FVIII-binding sites. These interactions follow the canonical binding mode providing major binding energy, and additional weak interactions are contributed by adjacent CR domains. A representative CR doublet was shown to have multiple contact sites on FVIII. CONCLUSIONS: FVIII and LRP1 interact via formation of multiple complex contacts involving both canonical and non-canonical binding combinations. We propose that FVIII-LRP1 interaction occurs via switching such alternative binding combinations in a dynamic mode, and that this mechanism is relevant to other ligand interactions of the low-density lipoprotein receptor family members including LRP1.

Molecular chaperone RAP interacts with LRP1 in a dynamic bivalent mode and enhances folding of ligand-binding regions of other LDLR family receptors.

The low-density lipoprotein receptor (LDLR) family of receptors are cell-surface receptors that internalize numerous ligands and play crucial role in various processes, such as lipoprotein metabolism, hemostasis, fetal development, etc. Previously, receptor-associated protein (RAP) was described as a molecular chaperone for LDLR-related protein 1 (LRP1), a prominent member of the LDLR family. We aimed to verify this role of RAP for LRP1 and two other LDLR family receptors, LDLR and vLDLR, and to investigate the mechanisms of respective interactions using a cell culture model system, purified system, and in silico modelling. Upon coexpression of RAP with clusters of the ligand-binding complement repeats (CRs) of the receptors in secreted form in insect cells culture, the isolated proteins had increased yield, enhanced folding, and improved binding properties compared with proteins expressed without RAP, as determined by circular dichroism and surface plasmon resonance. Within LRP1 CR-clusters II and IV, we identified multiple sites comprised of adjacent CR doublets, which provide alternative bivalent binding combinations with specific pairs of lysines on RAP. Mutational analysis of these lysines within each of isolated RAP D1/D2 and D3 domains having high affinity to LRP1 and of conserved tryptophans on selected CR-doublets of LRP1, as well as in silico docking of a model LRP1 CR-triplet with RAP, indicated a universal role for these residues in interaction of RAP and LRP1. Consequently, we propose a new model of RAP interaction with LDLR family receptors based on switching of the bivalent contacts between molecules over time in a dynamic mode.

Characterization of protein unable to bind von Willebrand factor in recombinant factor VIII products.

BACKGROUND: Therapeutic products with coagulation factor VIII (FVIII) have a wide range of specific activities, implying presence of protein with altered structure. Previous studies showed that recombinant FVIII products (rFVIII) contain a fraction (FVIIIFT ) unable to bind von Willebrand factor (VWF) and reported to lack activity. Because of loss of function(s), FVIIIFT can be defined as a product-related impurity, whose properties and levels in rFVIII products should be investigated. OBJECTIVE: To isolate and characterize the FVIIIFT fraction in rFVIII products. METHODS: Protein fractions unable (FVIIIFT ) and able (FVIIIEL ) to bind VWF were isolated from rFVIII products using immobilized VWF affinity chromatography (IVAC) and characterized by gel electrophoresis, immunoblotting, FVIII activity test, surface plasmon resonance, mass spectrometry, and for plasma clearance in mice. RESULTS AND CONCLUSIONS: A robust IVAC methodology was developed and applied for analysis of 10 rFVIII products marketed in the United States. FVIIIFT was found at various contents (0.4%-21.5%) in all products. Compared with FVIIIEL , FVIIIFT had similar patterns of polypeptide bands by gel electrophoresis, but lower functional activity. In several representative products, FVIIIFT was found to have reduced sulfation at Tyr1680, important for VWF binding, decreased interaction with a low-density lipoprotein receptor-related protein 1 fragment, and faster plasma clearance in mice. These findings provide basic characterization of FVIIIFT and demonstrate a potential for IVAC to control this impurity in rFVIII products to improve their efficacy in therapy of hemophilia A.

An extracellular histidine-containing motif in the zinc transporter ZIP4 plays a role in zinc sensing and zinc-induced endocytosis in mammalian cells.

Zinc is an essential trace element that serves as a cofactor for enzymes in critical biochemical processes and also plays a structural role in numerous proteins. Zinc transporter ZIP4 (ZIP4) is a zinc importer required for dietary zinc uptake in the intestine and other cell types. Studies in cultured cells have reported that zinc stimulates the endocytosis of plasma membrane-localized ZIP4 protein, resulting in reduced cellular zinc uptake. Thus, zinc-regulated trafficking of ZIP4 is a key means for regulating cellular zinc homeostasis, but the underlying mechanisms are not well understood. In this study, we used mutational analysis, immunoblotting, HEK293 cells, and immunofluorescence microscopy to identify a histidine-containing motif (398HTH) in the first extracellular loop that is required for high sensitivity to low zinc concentrations in a zinc-induced endocytic response of mouse ZIP4 (mZIP4). Moreover, using synthetic peptides with selective substitutions and truncated mZIP4 variants, we provide evidence that histidine residues in this motif coordinate a zinc ion in mZIP4 homodimers at the plasma membrane. These findings suggest that 398HTH is an important zinc-sensing motif for eliciting high-affinity zinc-stimulated endocytosis of mZIP4 and provide insight into cellular mechanisms for regulating cellular zinc homeostasis in mammalian cells.

Elesclomol restores mitochondrial function in genetic models of copper deficiency.

Copper is an essential cofactor of cytochrome c oxidase (CcO), the terminal enzyme of the mitochondrial respiratory chain. Inherited loss-of-function mutations in several genes encoding proteins required for copper delivery to CcO result in diminished CcO activity and severe pathologic conditions in affected infants. Copper supplementation restores CcO function in patient cells with mutations in two of these genes, COA6 and SCO2, suggesting a potential therapeutic approach. However, direct copper supplementation has not been therapeutically effective in human patients, underscoring the need to identify highly efficient copper transporting pharmacological agents. By using a candidate-based approach, we identified an investigational anticancer drug, elesclomol (ES), that rescues respiratory defects of COA6-deficient yeast cells by increasing mitochondrial copper content and restoring CcO activity. ES also rescues respiratory defects in other yeast mutants of copper metabolism, suggesting a broader applicability. Low nanomolar concentrations of ES reinstate copper-containing subunits of CcO in a zebrafish model of copper deficiency and in a series of copper-deficient mammalian cells, including those derived from a patient with SCO2 mutations. These findings reveal that ES can restore intracellular copper homeostasis by mimicking the function of missing transporters and chaperones of copper, and may have potential in treating human disorders of copper metabolism.

Organ-specific regulation of ATP7A abundance is coordinated with systemic copper homeostasis.

Copper (Cu) is an essential cofactor for various enzymatic activities including mitochondrial electron transport, iron mobilization, and peptide hormone maturation. Consequently, Cu dysregulation is associated with fatal neonatal disease, liver and cardiac dysfunction, and anemia. While the Cu transporter ATP7A plays a major role in both intestinal Cu mobilization to the periphery and prevention of Cu over-accumulation, it is unclear how regulation of ATP7A contributes to Cu homeostasis in response to systemic Cu fluctuation. Here we show, using Cu-deficient mouse models, that steady-state levels of ATP7A are lower in peripheral tissues (including the heart, spleen, and liver) under Cu deficiency and that subcutaneous administration of Cu to these animals restore normal ATP7A levels in these tissues. Strikingly, ATP7A in the intestine is regulated in the opposite manner - low systemic Cu increases ATP7A while subcutaneous Cu administration decreases ATP7A suggesting that intestine-specific non-autonomous regulation of ATP7A abundance may serve as a key homeostatic control for Cu export into the circulation. Our results support a systemic model for how a single transporter can be inversely regulated in a tissue-specific manner to maintain organismal Cu homeostasis.

The Intestinal Copper Exporter CUA-1 Is Required for Systemic Copper Homeostasis in Caenorhabditis elegans.

Copper plays key catalytic and regulatory roles in biochemical processes essential for normal growth, development, and health. Defects in copper metabolism cause Menkes and Wilson's disease, myeloneuropathy, and cardiovascular disease and are associated with other pathophysiological states. Consequently, it is critical to understand the mechanisms by which organisms control the acquisition, distribution, and utilization of copper. The intestinal enterocyte is a key regulatory point for copper absorption into the body; however, the mechanisms by which intestinal cells transport copper to maintain organismal copper homeostasis are poorly understood. Here, we identify a mechanism by which organismal copper homeostasis is maintained by intestinal copper exporter trafficking that is coordinated with extraintestinal copper levels in Caenorhabditis elegans Specifically, we show that CUA-1, the C. elegans homolog of ATP7A/B, localizes to lysosome-like organelles (gut granules) in the intestine under copper overload conditions for copper detoxification, whereas copper deficiency results in a redistribution of CUA-1 to basolateral membranes for copper efflux to peripheral tissues. Worms defective in gut granule biogenesis exhibit defects in copper sequestration and increased susceptibility to toxic copper levels. Interestingly, however, a splice isoform CUA-1.2 that lacks a portion of the N-terminal domain is targeted constitutively to the basolateral membrane irrespective of dietary copper concentration. Our studies establish that CUA-1 is a key intestinal copper exporter and that its trafficking is regulated to maintain systemic copper homeostasis. C. elegans could therefore be exploited as a whole-animal model system to study regulation of intra- and intercellular copper trafficking pathways.

Discovery of novel HCV polymerase inhibitors using pharmacophore-based virtual screening.

We report the use of pharmacophore-based virtual screening as an efficient tool for the discovery of novel HCV polymerase inhibitors. A three-dimensional pharmacophore model for the HCV-796 binding site, NNI site IV inhibitor, to the enzyme was built by means of the structure-based focusing module in Cerius2 program. Using these models as a query for virtual screening, we produced a successful example of using pharmacophore-based virtual screening to identify novel compounds with HCV replicon assay through inhibition of HCV polymerization. Among the hit compounds, compounds 1 and 2 showed 56% and 48% inhibition of NS5B polymerization activity at 20 µM, respectively. In addition, compound 1 also exhibited replicon activity with EC(50) value of 2.16 µM. Following up the initial hit, we obtained derivatives of compound 1 and evaluated polymerization inhibition activity and HCV replicon assay. These results provide information necessary for the development of more potent NS5B inhibitors.

Design and efficient production of bovine enterokinase light chain with higher specificity in E. coli.

Enterokinase light chain (EKL) is a serine protease that recognizes Asp-Asp-Asp-Asp-Lys (D(4)K) sequence and cleaves the C-terminal peptide bond of the lysine residue. The utility of EKL as a site-specific cleavage enzyme is hampered by sporadic cleavage at other sites than the canonical D(4)K recognition sequence. In order to produce more site-specific EKL, we have generated several EKL mutants in E. coli with substitutions at Tyr174 and Lys99 using PDI (protein disulfide isomerase) fusion system. Substitution of Tyr174 by basic residues confers higher specificity on EKL. The production of EKL with higher specificity could widen the utility of EKL as a site-specific cleavage enzyme to produce various recombinant proteins with therapeutic or industrial values.

Structural modeling of V299L and E459K Bcr-Abl mutation, and sequential therapy of tyrosine kinase inhibitors for the compound mutations.

Sequential treatment with different tyrosine kinase inhibitors (TKIs) is one of the strategies for handling chronic myeloid leukemia (CML) in which dynamic change in Bcr-Abl kinase domain mutation is often an obstacle faced during TKI therapy. Here we report successful sequential therapy with different TKIs for the CML patient harboring V299L and E459K compound mutations. Molecular monitoring including quantitative analysis of BCR-ABL transcript level and mutation analysis were performed regularly for successful treatment. Additionally a drug-target complex was structurally modeled to investigate influence of amino acid substitutions on drug resistance, and to choose alternative TKI in sequential therapy, suggesting protein structural modeling can be useful approach in selecting alternative TKIs.