Structure and mechanism of the mammalian fructose transporter GLUT5.

The altered activity of the fructose transporter GLUT5, an isoform of the facilitated-diffusion glucose transporter family, has been linked to disorders such as type 2 diabetes and obesity. GLUT5 is also overexpressed in certain tumour cells, and inhibitors are potential drugs for these conditions. Here we describe the crystal structures of GLUT5 from Rattus norvegicus and Bos taurus in open outward- and open inward-facing conformations, respectively. GLUT5 has a major facilitator superfamily fold like other homologous monosaccharide transporters. On the basis of a comparison of the inward-facing structures of GLUT5 and human GLUT1, a ubiquitous glucose transporter, we show that a single point mutation is enough to switch the substrate-binding preference of GLUT5 from fructose to glucose. A comparison of the substrate-free structures of GLUT5 with occluded substrate-bound structures of Escherichia coli XylE suggests that, in addition to global rocker-switch-like re-orientation of the bundles, local asymmetric rearrangements of carboxy-terminal transmembrane bundle helices TM7 and TM10 underlie a 'gated-pore' transport mechanism in such monosaccharide transporters.

Prdm16 is crucial for progression of the multipolar phase during neural differentiation of the developing neocortex.

The precise control of neuronal migration and morphological changes during differentiation is essential for neocortical development. We hypothesized that the transition of progenitors through progressive stages of differentiation involves dynamic changes in levels of mitochondrial reactive oxygen species (mtROS), depending on cell requirements. We found that progenitors had higher levels of mtROS, but that these levels were significantly decreased with differentiation. The Prdm16 gene was identified as a candidate modulator of mtROS using microarray analysis, and was specifically expressed by progenitors in the ventricular zone. However, Prdm16 expression declined during the transition into NeuroD1-positive multipolar cells. Subsequently, repression of Prdm16 expression by NeuroD1 on the periphery of ventricular zone was crucial for appropriate progression of the multipolar phase and was required for normal cellular development. Furthermore, time-lapse imaging experiments revealed abnormal migration and morphological changes in Prdm16-overexpressing and -knockdown cells. Reporter assays and mtROS determinations demonstrated that PGC1α is a major downstream effector of Prdm16 and NeuroD1, and is required for regulation of the multipolar phase and characteristic modes of migration. Taken together, these data suggest that Prdm16 plays an important role in dynamic cellular redox changes in developing neocortex during neural differentiation.

Single-Dose Cisplatin Pre-Treatment Enhances Efficacy of ROBO1-Targeted Radioimmunotherapy.

We previously reported that radioimmunotherapy (RIT) using 90Y-labeled anti-ROBO1 IgG (90Y-B5209B) achieved significant anti-tumor effects against small-cell lung cancer (SCLC) xenografts. However, subsequent tumor regrowth suggested the necessity for more effective therapy. Here, we evaluated the efficacy of combination 90Y-B5209B and cisplatin therapy in NCI-H69 SCLC xenograft mice. Mice were divided into four therapeutic groups: saline, cisplatin only, RIT only, or combination therapy. Either saline or cisplatin was administered by injection one day prior to the administration of either saline or 90Y-B5209B. Tumor volume, body weight, and blood cell counts were monitored. The pathological analysis was performed on day seven post injection of 90Y-B5209B. The survival duration of the combination therapy group was significantly longer than that of the group treated with RIT alone. No significant survival benefit was observed following the isolated administration of cisplatin (relative to saline). Pathological changes following combination therapy were more significant than those following the isolated administration of RIT. Although combination therapy was associated with an increase of several adverse effects such as weight loss and pancytopenia, these were transient. Thus, cisplatin pre-treatment can potentially enhance the efficacy of 90Y-B5209B, making it a promising therapeutic strategy for SCLC.

Structural behavior of keratin-associated protein 8.1 in human hair as revealed by a monoclonal antibody.

Keratin-associated protein 8.1 (KAP8.1) is a hair protein whose structure, biochemical roles, and protein distribution patterns have not been well characterized. In this study, we generated a monoclonal antibody against human KAP8.1 to analyze the protein's roles and distribution in the human hair shaft. Using this antibody, we revealed that KAP8.1 was predominantly expressed in discrete regions of the keratinizing zone of the hair shaft cortex. The protein expression patterns paralleled the distribution of KAP8.1 mRNA and suggested that KAP8.1 plays a role associated with cells to control hair curvature. Cross-reactivity among species and epitope analysis indicated that the monoclonal antibody recognized a linear epitope shared among human, mouse, and sheep KAP8.1. The antibody failed to interact with sheep KAP8.1 in native conformation, suggesting that structural features of KAP8.1 vary among species.

G-protein-coupled receptor inactivation by an allosteric inverse-agonist antibody.

G-protein-coupled receptors are the largest class of cell-surface receptors, and these membrane proteins exist in equilibrium between inactive and active states. Conformational changes induced by extracellular ligands binding to G-protein-coupled receptors result in a cellular response through the activation of G proteins. The A(2A) adenosine receptor (A(2A)AR) is responsible for regulating blood flow to the cardiac muscle and is important in the regulation of glutamate and dopamine release in the brain. Here we report the raising of a mouse monoclonal antibody against human A(2A)AR that prevents agonist but not antagonist binding to the extracellular ligand-binding pocket, and describe the structure of A(2A)AR in complex with the antibody Fab fragment (Fab2838). This structure reveals that Fab2838 recognizes the intracellular surface of A(2A)AR and that its complementarity-determining region, CDR-H3, penetrates into the receptor. CDR-H3 is located in a similar position to the G-protein carboxy-terminal fragment in the active opsin structure and to CDR-3 of the nanobody in the active ß(2)-adrenergic receptor structure, but locks A(2A)AR in an inactive conformation. These results suggest a new strategy to modulate the activity of G-protein-coupled receptors.

Suppression of Slit2/Robo1 mediated HUVEC migration by Robo4.

Slit proteins and their receptors, the Roundabout (Robo) family, are known to have a pivotal role in the vascular system. Slit2/Robo1 regulates the migration of human umbilical vein endothelial cells (HUVECs) and tumor-associated endothelial cells. Robo4, the endothelial-specific Robo, is also considered to be involved in vascular cell migration. However, the Slit/Robo signaling pathway is still unclear. Using a Boyden chamber assay, we found that Slit2 induces the migration of HUVECs under a Robo4 knockdown condition. This effect disappeared in Robo1 knockdown cells. The co-existence of the N-terminal extracellular portion of Robo1 blocked the Slit2-evoked migration of HUVECs, while that of Robo4 caused no effect. These results show that the Slit2 signal is transduced through Robo1, while the negative regulation of Robo4 is an intracellular event. Targeted proteomics using an anti-Robo1 monoclonal antibody identified CdGAP, an adhesion-localized Rac1-and Cdc42-specific GTPase activating protein, as a candidate for Slit2/Robo1 signaling. Robo1 and CdGAP were co-immunoprecipitated from CHO cells co-transfected with Robo1 and CdGAP genes. These results suggest that Slit2/Robo1 binding exerts an effect on cell migration, which is negatively regulated by Robo4, and Robo1 may function by interacting with CdGAP in HUVECs.

G protein-dependent basal and evoked endothelial cell vWF secretion.

von Willebrand factor (vWF) secretion by endothelial cells (ECs) is essential for hemostasis and thrombosis; however, the molecular mechanisms are poorly understood. Interestingly, we observed increased bleeding in EC-Gα13(-/-);Gα12(-/-) mice that could be normalized by infusion of human vWF. Blood from Gα12(-/-) mice exhibited significantly reduced vWF levels but normal vWF multimers and impaired laser-induced thrombus formation, indicating that Gα12 plays a prominent role in EC vWF secretion required for hemostasis and thrombosis. In isolated buffer-perfused mouse lungs, basal vWF levels were significantly reduced in Gα12(-/-), whereas thrombin-induced vWF secretion was defective in both EC-Gαq(-/-);Gα11(-/-) and Gα12(-/-) mice. Using siRNA in cultured human umbilical vein ECs and human pulmonary artery ECs, depletion of Gα12 and soluble N-ethylmaleimide-sensitive-fusion factor attachment protein α (α-SNAP), but not Gα13, inhibited both basal and thrombin-induced vWF secretion, whereas overexpression of activated Gα12 promoted vWF secretion. In Gαq, p115 RhoGEF, and RhoA-depleted human umbilical vein ECs, thrombin-induced vWF secretion was reduced by 40%, whereas basal secretion was unchanged. Finally, in vitro binding assays revealed that Gα12 N-terminal residues 10-15 mediated the binding of Gα12 to α-SNAP, and an engineered α-SNAP binding-domain minigene peptide blocked basal and evoked vWF secretion. Discovery of obligatory and complementary roles of Gα12 and Gαq/11 in basal vs evoked EC vWF secretion may provide promising new therapeutic strategies for treatment of thrombotic disease.

Kinetic exclusion assay of monoclonal antibody affinity to the membrane protein Roundabout 1 displayed on baculovirus.

The reliable assessment of monoclonal antibody (mAb) affinity against membrane proteins in vivo is a major issue in the development of cancer therapeutics. We describe here a simple and highly sensitive method for the evaluation of mAbs against membrane proteins by means of a kinetic exclusion assay (KinExA) in combination with our previously developed membrane protein display system using budded baculovirus (BV). In our BV display system, the membrane proteins are displayed on the viral surface in their native form. The BVs on which the liver cancer antigen Roundabout 1 (Robo1) was displayed were adsorbed onto magnetic beads without fixative (BV beads). The dissociation constant (Kd, ∼10(-11) M) that was measured on the Robo1 expressed BV beads correlated well with the value from a whole cell assay (the coefficient of determination, R(2) = 0.998) but not with the value for the soluble extracellular domains of Robo1 (R(2) = 0.834). These results suggest that the BV-KinExA method described here provides a suitably accurate Kd evaluation of mAbs against proteins on the cell surface.

Generation of antibodies against membrane proteins.

The monoclonal antibody has become an important therapeutic in the treatment of both hematological malignancies and solid tumors. The recent success of antibody-drug conjugates (ADCs) has broadened the extent of the potential target molecules in cancer immunotherapy. As a result, even molecules of low abundance have become targets for cytotoxic reagents. The multi-pass membrane proteins are an emerging target for the next generation antibody therapeutics. One outstanding challenge is the difficulty in preparing a sufficient amount of these membrane proteins so as to be able to generate the functional antibody. We have pursued the expression of various membrane proteins on the baculovirus particle and the utilization of displayed protein for immunization. The strong antigenicity of the virus acts either as a friend or foe in the making of an efficient antibody against an immunologically tolerant antigen. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody.

Epigenetically coordinated GATA2 binding is necessary for endothelium-specific endomucin expression.

GATA2 is well recognized as a key transcription factor and regulator of cell-type specificity and differentiation. Here, we carried out comparative chromatin immunoprecipitation with comprehensive sequencing (ChIP-seq) to determine genome-wide occupancy of GATA2 in endothelial cells and erythroids, and compared the occupancy to the respective gene expression profile in each cell type. Although GATA2 was commonly expressed in both cell types, different GATA2 bindings and distinct cell-specific gene expressions were observed. By using the ChIP-seq with epigenetic histone modifications and chromatin conformation capture assays; we elucidated the mechanistic regulation of endothelial-specific GATA2-mediated endomucin gene expression, that was regulated by the endothelial-specific chromatin loop with a GATA2-associated distal enhancer and core promoter. Knockdown of endomucin markedly attenuated endothelial cell growth, migration and tube formation. Moreover, abrogation of GATA2 in endothelium demonstrated not only a reduction of endothelial-specific markers, but also induction of mesenchymal transition promoting gene expression. Our findings provide new insights into the correlation of endothelial-expressed GATA2 binding, epigenetic modification, and the determination of endothelial cell specificity.

Analysis of the subcellular localization of the human histone methyltransferase SETDB1.

SET domain, bifurcated 1 (SETDB1) is a histone methyltransferase that methylates lysine 9 on histone H3. Although it is important to know the localization of proteins to elucidate their physiological function, little is known of the subcellular localization of human SETDB1. In the present study, to investigate the subcellular localization of hSETDB1, we established a human cell line constitutively expressing enhanced green fluorescent protein fused to hSETDB1. We then generated a monoclonal antibody against the hSETDB1 protein. Expression of both exogenous and endogenous hSETDB1 was observed mainly in the cytoplasm of various human cell lines. Combined treatment with the nuclear export inhibitor leptomycin B and the proteasome inhibitor MG132 led to the accumulation of hSETDB1 in the nucleus. These findings suggest that hSETDB1, localized in the nucleus, might undergo degradation by the proteasome and be exported to the cytosol, resulting in its detection mainly in the cytosol.

Phosphatidylinositol 3-phosphatase myotubularin-related protein 6 (MTMR6) is regulated by small GTPase Rab1B in the early secretory and autophagic pathways.

A large family of myotubularin phosphatases dephosphorylates phosphatidylinositol 3-phosphate and phosphatidylinositol 3,5-bisphosphate, which are known to play important roles in vesicular trafficking and autophagy. The family is composed of 16 members, and understanding their regulatory mechanisms is important to understand their functions and related genetic diseases. We prepared anti-myotubularin-related protein 6 (MTMR6) monoclonal antibody and used it to study the regulatory mechanism of MTMR6. Endogenous MTMR6 was present in the cytoplasm and was condensed in the perinuclear region in a microtubule-dependent manner. MTMR6 preferentially interacted with GDP-bound Rab1B via the GRAM domain and partly overlapped with Rab1B in the pericentrosomal and peri-Golgi regions in normal rat kidney cells. Overexpression of GDP-bound Rab1B and the reduction of Rab1B disrupted the localization of MTMR6, suggesting that Rab1B regulates the localization of MTMR6. The reduction of MTMR6 accelerated the transport of vesicular stomatitis virus glycoprotein in which Rab1B is involved. Furthermore, reduction of MTMR6 or Rab1B inhibited the formation of the tubular omegasome that is induced by overexpression of DFCP1 in autophagy. Our results indicate that the cellular localization of MTMR6 is regulated by Rab1B in the early secretory and autophagic pathways. We propose a new regulatory mechanism of myotubularin phosphatase by the small GTPase Rab1B.

Identification of Wilms' tumor 1-associating protein complex and its role in alternative splicing and the cell cycle.

Wilms' tumor 1-associating protein (WTAP) is a putative splicing regulator that is thought to be required for cell cycle progression through the stabilization of cyclin A2 mRNA and mammalian early embryo development. To further understand how WTAP acts in the context of the cellular machinery, we identified its interacting proteins in human umbilical vein endothelial cells and HeLa cells using shotgun proteomics. Here we show that WTAP forms a novel protein complex including Hakai, Virilizer homolog, KIAA0853, RBM15, the arginine/serine-rich domain-containing proteins BCLAF1 and THRAP3, and certain general splicing regulators, most of which have reported roles in post-transcriptional regulation. The depletion of these respective components of the complex resulted in reduced cell proliferation along with G2/M accumulation. Double knockdown of the serine/arginine-rich (SR)-like proteins BCLAF1 and THRAP3 by siRNA resulted in a decrease in the nuclear speckle localization of WTAP, whereas the nuclear speckles were intact. Furthermore, we found that the WTAP complex regulates alternative splicing of the WTAP pre-mRNA by promoting the production of a truncated isoform, leading to a change in WTAP protein expression. Collectively, these findings show that the WTAP complex is a novel component of the RNA processing machinery, implying an important role in both posttranscriptional control and cell cycle regulation.

Novel role of copper transport protein antioxidant-1 in neointimal formation after vascular injury.

OBJECTIVE: Vascular smooth muscle cell (VSMC) migration is critically important for neointimal formation after vascular injury and atherosclerosis lesion formation. Copper (Cu) chelator inhibits neointimal formation, and we previously demonstrated that Cu transport protein antioxidant-1 (Atox1) is involved in Cu-induced cell growth. However, role of Atox1 in VSMC migration and neointimal formation after vascular injury is unknown. APPROACH AND RESULTS: Here, we show that Atox1 expression is upregulated in injured vessel, and it is colocalized with the Cu transporter ATP7A, one of the downstream targets of Atox1, mainly in neointimal VSMCs at day 14 after wire injury. Atox1(-/-) mice show inhibition of neointimal formation and extracellular matrix expansion, which is associated with a decreased VSMCs accumulation within neointima and lysyl oxidase activity. Mechanistically, in cultured VSMC, Atox1 depletion with siRNA inhibits platelet-derived growth factor-induced Cu-dependent VSMC migration by preventing translocation of ATP7A and small G protein Rac1 to the leading edge, as well as Cu- and Rac1-dependent lamellipodia formation. Furthermore, Atox1(-/-) mice show decreased perivascular macrophage infiltration in wire-injured vessels, as well as thioglycollate-induced peritoneal macrophage recruitment. CONCLUSIONS: Atox1 is involved in neointimal formation after vascular injury through promoting VSMC migration and inflammatory cell recruitment in injured vessels. Thus, Atox1 is a potential therapeutic target for VSMC migration and inflammation-related vascular diseases.

Inhibition of DNA damage-induced apoptosis through Cdc7-mediated stabilization of Tob.

BACKGROUND: Preventing unnecessary cell death is essential for DNA-damaged cells to carry out the DNA repair process. RESULTS: Cdc7 inhibits the Cul4-DDB1(Cdt2)-dependent Tob degradation. CONCLUSION: Cdc7 enables mild DNA-damaged cells to keep their viability by competing with the Tob degradation system. SIGNIFICANCE: Cells deal with moderate DNA damage not only by cessation of the cell cycle but also through direct mediated pro-survival signaling. Cells respond to DNA damage by activating alternate signaling pathways that induce proliferation arrest or apoptosis. The correct balance between these two pathways is important for maintaining genomic integrity and preventing unnecessary cell death. The mechanism by which DNA-damaged cells escape from apoptosis during DNA repair is poorly understood. We show that the DNA replication-initiating kinase Cdc7 actively prevents unnecessary death in DNA-damaged cells. In response to mild DNA damage, Tob levels increase through both a transcriptional mechanism and protein stabilization, resulting in inhibition of pro-apoptotic signaling. Cells lacking Cdc7 expression undergo apoptosis after mild DNA damage, where Cul4-DDB1(Cdt2) induces Tob ubiquitination and subsequent degradation. Cdc7 phosphorylates and interacts with Tob to inhibit the Cul4-DDB1(Cdt2)-dependent Tob degradation. Thus, Cdc7 defines an essential pro-survival signaling pathway by contributing to stabilization of Tob, thereby the viability of DNA-damaged cells being maintained.

Structure and mechanism of oxalate transporter OxlT in an oxalate-degrading bacterium in the gut microbiota.

An oxalate-degrading bacterium in the gut microbiota absorbs food-derived oxalate to use this as a carbon and energy source, thereby reducing the risk of kidney stone formation in host animals. The bacterial oxalate transporter OxlT selectively uptakes oxalate from the gut to bacterial cells with a strict discrimination from other nutrient carboxylates. Here, we present crystal structures of oxalate-bound and ligand-free OxlT in two distinct conformations, occluded and outward-facing states. The ligand-binding pocket contains basic residues that form salt bridges with oxalate while preventing the conformational switch to the occluded state without an acidic substrate. The occluded pocket can accommodate oxalate but not larger dicarboxylates, such as metabolic intermediates. The permeation pathways from the pocket are completely blocked by extensive interdomain interactions, which can be opened solely by a flip of a single side chain neighbouring the substrate. This study shows the structural basis underlying metabolic interactions enabling favourable symbiosis.

Single chain variable fragment against nicastrin inhibits the gamma-secretase activity.

Gamma-secretase is a membrane protein complex that catalyzes intramembrane proteolysis of a variety of substrates including the amyloid beta precursor protein of Alzheimer disease. Nicastrin (NCT), a single-pass membrane glycoprotein that harbors a large extracellular domain, is an essential component of the gamma-secretase complex. Here we report that overexpression of a single chain variable fragment (scFv) against NCT as an intrabody suppressed the gamma-secretase activity. Biochemical analyses revealed that the scFv disrupted the proper folding and the appropriate glycosyl maturation of the endogenous NCT, which are required for the stability of the gamma-secretase complex and the intrinsic proteolytic activity, respectively, implicating the dual role of NCT in the gamma-secretase complex. Our results also highlight the importance of the calnexin cycle in the functional maturation of the gamma-secretase complex. The engineered intrabodies may serve as rationally designed, molecular targeting tools for the discovery of novel actions of the membrane proteins.

AT2 receptor interacting protein 1 (ATIP1) mediates COX-2 induction by an AT2 receptor agonist in endothelial cells.

Angiotensin II (Ang II) type 2 receptor (AT2R) is one of the major components of the renin-angiotensin-aldosterone system. Nevertheless, the physiological role is not well defined compared to the understanding of the Ang II type 1 receptor (AT1R), which is a well characterized G-protein coupled receptor in the cardiovascular system. While the AT2R signaling pathway remains unclear, AT2 receptor interacting protein 1 (ATIP1) has been identified as a candidate molecule for interacting with the C-terminal region of AT2R. In this study, we investigated the ATIP1 dependent AT2R inducible genes in human umbilical vein endothelial cells (HUVECs). CGP42112A, an AT2R specific agonist, resulted in an upregulation of inflammatory genes in HUVECs, which were inhibited by knocking down ATIP1 with siRNA (siATIP1). Among them, we confirmed by quantitative PCR that the induction of COX-2 mRNA expression was significantly downregulated by siATIP1. COX-2 was also upregulated by Ang II stimulation. This upregulation was suppressed by treatment with the AT2R specific antagonist PD123319, which was not replicated by the AT1R antagonist telmisartan. These findings suggest that ATIP1 plays an important role in AT2R dependent inflammatory responses. This may provide a new approach to the development of cardio-protective drugs.

Generation of biparatopic antibody through two-step targeting of fragment antibodies on antigen using SpyTag and SpyCatcher.

Biparatopic fragment antibodies can overcome deficiencies in avidity of conventional antibody fragments. Here, we describe a technology for generating biparatopic antibodies through two-step targeting using a pair of polypeptides, SpyTag and SpyCatcher, that spontaneously react to form a covalent bond between antibody fragments. In this method, two antibody fragments, each targeting different epitopes of the antigen, are fused to SpyTag and to SpyCatcher. When the two polypeptides are serially added to the antigen, their proximity on the antigen results in covalent bond formation and generation of a biparatopic antibody. We validated the system with purified recombinant antigen. Results in antigen-overexpressing cells were promising although further optimization will be required. Because this strategy results in high-affinity targeting with a bipartite molecule that has considerably lower molecular weight than an antibody, this technology is potentially useful for diverse applications.