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Midkine (MK), a heparin-binding growth factor, is associated with the poor prognosis of the pediatric tumor, neuroblastoma. MK would be a druggable target as many studies showed inhibition of its function in various cancers suppressed tumor developments. To establish the therapy targeting MK, identification of its binding partners, and elucidation of its intracellular signaling are needed. It was reported that exogenous MK induced phosphorylation of ribosomal protein S6 (RPS6) downstream of mTOR signaling. Using RPS6 phosphorylation as a marker of MK response, we searched for MK reactive cell lines. We found that MK cell lines expressing less MK tended to respond better to MK. Next, using an MK reactive neuroblastoma cell line, MK-knocked down SH-SY5Y cells, we employed a proximity-dependent biotin identification method, which was invented to evaluate protein-protein interactions by biotinylation. We confirmed that secreted MK fused to the biotin ligase BioID2 (MK-BioID2) was able to biotinylate proteins from the cells. Biotinylated proteins were identified by liquid chromatography-mass spectrometry analyses. Twenty five proteins were found to be overlapped after three independent experiments, among which insulin-like growth binding protein 2 (IGFBP2) was further analyzed. IGFBP2 was indeed detected with immunoblotting after streptavidin pull down of MK-BioID2 labeled cell extract of MK-knocked down SH-SY5Y cells. Our study suggests that the BioID2 method is useful to identify binding partners of growth factors., This is an Open Access article distributed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License. To view the details of this license, please visit (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Midkine (MK) is a neurotrophic factor that participates in the embryonic central nervous system (CNS) development and neural stem cell regulation, interacting with sulfated glycosaminoglycans (GAGs). Chondroitin sulfate (CS) is the natural ligand in the CNS. In this work, we describe the interactions between a library of synthetic models of CS-types and mimics. We did a structural study of this library by NMR and MD (Molecular Dynamics), concluding that the basic shape is controlled by similar geometry of the glycosidic linkages. Their 3D structures are a helix with four residues per turn, almost linear. We have studied the tetrasaccharide-midkine complexes by ligand observed NMR techniques and concluded that the shape of the ligands does not change upon binding. The ligand orientation into the complex is very variable. It is placed inside the central cavity of MK formed by the two structured beta-sheets domains linked by an intrinsically disordered region (IDR). Docking analysis confirmed the participation of aromatics residues from MK completed with electrostatic interactions. Finally, we test the biological activity by increasing the MK expression using CS tetrasaccharides and their capacity in enhancing the growth stimulation effect of MK in NIH3T3 cells.

type:text, The small roundworm Caenorhabditis elegans employs two strategies, termed pirouette and weathervane, which are closely related to the internal representation of chemical gradients parallel and perpendicular to the travelling direction, respectively, to perform chemotaxis. These gradients must be calculated from the chemical information obtained at a single point, because the sensory neurons are located close to each other at the nose tip. To formulate the relationship between this sensory input and internal representations of the chemical gradient, this study proposes a simple computational model derived from the directional decomposition of the chemical concentration at the nose tip that can generate internal representations of the chemical gradient. The ability of the computational model was verified by using a chemotaxis simulator that can simulate the body motions of pirouette and weathervane, which confirmed that the computational model enables the conversion of the sensory input and head-bending angles into both types of gradients with high correlations of approximately r > 0.90 (p < 0.01) with the true gradients. In addition, the chemotaxis index of the model was 0.64, which is slightly higher than that in the actual animal (0.57). In addition, simulation using a connectome-based neural network model confirmed that the proposed computational model is implementable in the actual network structure., This work was supported by JSPS KAKENHI Grant Number 15H03950 to T.T and MEXT KAKENHI Grant Numbers 20115010 to T.T. and 20115002 to Y.I.

The small roundworm Caenorhabditis elegans employs two strategies, termed pirouette and weathervane, which are closely related to the internal representation of chemical gradients parallel and perpendicular to the travelling direction, respectively, to perform chemotaxis. These gradients must be calculated from the chemical information obtained at a single point, because the sensory neurons are located close to each other at the nose tip. To formulate the relationship between this sensory input and internal representations of the chemical gradient, this study proposes a simple computational model derived from the directional decomposition of the chemical concentration at the nose tip that can generate internal representations of the chemical gradient. The ability of the computational model was verified by using a chemotaxis simulator that can simulate the body motions of pirouette and weathervane, which confirmed that the computational model enables the conversion of the sensory input and head-bending angles into both types of gradients with high correlations of approximately r > 0.90 (p < 0.01) with the true gradients. In addition, the chemotaxis index of the model was 0.64, which is slightly higher than that in the actual animal (0.57). In addition, simulation using a connectome-based neural network model confirmed that the proposed computational model is implementable in the actual network structure., This work was supported by JSPS KAKENHI Grant Number 15H03950 to T.T and MEXT KAKENHI Grant Numbers 20115010 to T.T. and 20115002 to Y.I.

The small roundworm Caenorhabditis elegans employs two strategies, termed pirouette and weathervane, which are closely related to the internal representation of chemical gradients parallel and perpendicular to the travelling direction, respectively, to perform chemotaxis. These gradients must be calculated from the chemical information obtained at a single point, because the sensory neurons are located close to each other at the nose tip. To formulate the relationship between this sensory input and internal representations of the chemical gradient, this study proposes a simple computational model derived from the directional decomposition of the chemical concentration at the nose tip that can generate internal representations of the chemical gradient. The ability of the computational model was verified by using a chemotaxis simulator that can simulate the body motions of pirouette and weathervane, which confirmed that the computational model enables the conversion of the sensory input and head-bending angles into both types of gradients with high correlations of approximately r > 0.90 (p < 0.01) with the true gradients. In addition, the chemotaxis index of the model was 0.64, which is slightly higher than that in the actual animal (0.57). In addition, simulation using a connectome-based neural network model confirmed that the proposed computational model is implementable in the actual network structure., This work was supported by JSPS KAKENHI Grant Number 15H03950 to T.T and MEXT KAKENHI Grant Numbers 20115010 to T.T. and 20115002 to Y.I.

Protein production within the secretory pathway is accomplished by complex but organized processes. Here, we demonstrate that the growth factor midkine interacts with LDL receptor-related protein 1 (LRP1) at high affinity (K d value, 2.7 nM) not only at the cell surface but also within the secretory pathway during biosynthesis. The latter premature ligand-receptor interaction resulted in aggregate formation and consequently suppressed midkine secretion and LRP1 maturation. We utilized an endoplasmic reticulum (ER) retrieval signal and an LRP1 fragment, which strongly bound to midkine and the LRP1-specialized chaperone receptor-associated protein (RAP), to construct an ER trapper. The ER trapper efficiently trapped midkine and RAP and mimicked the premature ligand-receptor interaction, i.e. suppressed maturation of the ligand and receptor. The ER trapper also diminished the inhibitory function of LRP1 on platelet-derived growth factor-mediated cell migration. Complementary to these results, an increased expression of RAP was closely associated with midkine expression in human colorectal carcinomas (33 of 39 cases examined). Our results suggest that the premature ligand-receptor interaction plays a role in protein production within the secretory pathway. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

The growth factor midkine (MK) is highly associated with cancer progression. Knockdown of MK expression strikingly suppresses tumor growth in nude mice. Thus, MK is a candidate target for cancer treatment. LDL-receptor-related protein 1 (LRP1) is a receptor for MK. We found that among the four ligand-binding domains of LRP1, the N-terminal half of the second domain (designated as MK-TRAP) had the strongest affinity to MK. MK-TRAP bound to MK, but not to HB-GAM/ pleiotrophin, basic fibroblast growth factor or platelet-derived growth factor (PDGF)-BB. Exogenous MK-TRAP inhibited the binding between MK and LRP1. G401 cells that transiently or stably overexpress MK-TRAP showed decreased cell growth in monolayer culture and reduced colony formation in soft agar, which could be rescued by exogenous MK administration. MK-TRAP collected from conditioned medium also inhibited anchorage-independent growth of G401 cells and CMT-93 cells. Anti-MK antibody also inhibited the anchorage-independent growth. CMT-93 cells stably expressing MK-TRAP formed smaller tumors in a xenograft nude mouse model than control cells. Moreover, GST-RAP, a potent inhibitor of LRP1, inhibited the anchorage-independent growth of control G401 cells but not that of MK-TRAP stable transformants. Collectively, these data demonstrate a crucial role of MK-LRP1 signaling in anchorage-independent cell growth.

The small roundworm Caenorhabditis elegans employs two strategies, termed pirouette and weathervane, which are closely related to the internal representation of chemical gradients parallel and perpendicular to the travelling direction, respectively, to perform chemotaxis. These gradients must be calculated from the chemical information obtained at a single point, because the sensory neurons are located close to each other at the nose tip. To formulate the relationship between this sensory input and internal representations of the chemical gradient, this study proposes a simple computational model derived from the directional decomposition of the chemical concentration at the nose tip that can generate internal representations of the chemical gradient. The ability of the computational model was verified by using a chemotaxis simulator that can simulate the body motions of pirouette and weathervane, which confirmed that the computational model enables the conversion of the sensory input and head-bending angles into both types of gradients with high correlations of approximately r > 0.90 (p < 0.01) with the true gradients. In addition, the chemotaxis index of the model was 0.64, which is slightly higher than that in the actual animal (0.57). In addition, simulation using a connectome-based neural network model confirmed that the proposed computational model is implementable in the actual network structure., This work was supported by JSPS KAKENHI Grant Number 15H03950 to T.T and MEXT KAKENHI Grant Numbers 20115010 to T.T. and 20115002 to Y.I.