IBS Institute for Basic Science
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Reconfiguration of the proteasomeduring chaperone-mediated assembly.
Reconfiguration of the proteasomeduring chaperone-mediated assembly.

    The proteasomal ATPase ring, comprising Rpt1–Rpt6, associates with the heptameric α-ring of the proteasome core particle (CP) in the mature proteasome, with the Rpt carboxy-terminal tails inserting into pockets of the α-ring1, 2, 3, 4. Rpt ring assembly is mediated by four chaperones, each binding a distinct Rpt subunit5, 6, 7, 8, 9, 10. Here we report that the base subassembly of the Saccharomyces cerevisiae proteasome, which includes the Rpt ring, forms a high-affinity complex with the CP. This complex is subject to active dissociation by the chaperones Hsm3, Nas6 and Rpn14. Chaperone-mediated dissociation was abrogated by a non-hydrolysable ATP analogue, indicating that chaperone action is coupled to nucleotide hydrolysis by the Rpt ring. Unexpectedly, synthetic Rpt tail peptides bound α-pockets with poor specificity, except for Rpt6, which uniquely bound the α2/α3-pocket. Although the Rpt6 tail is not visualized within an α-pocket in mature proteasomes2, 3, 4, it inserts into the α2/α3-pocket in the base–CP complex and is important for complex formation. Thus, the Rpt–CP interface is reconfigured when the lid complex joins the nascent proteasome to form the mature holoenzyme.

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Structural basis for LAR-RPTP/Slitrk complex-mediated synaptic adhesion.
Structural basis for LAR-RPTP/Slitrk complex-mediated synaptic adhesion.

    Synaptic adhesion molecules orchestrate synaptogenesis. The presynaptic leukocyte common antigen-related receptor protein tyrosine phosphatases (LAR-RPTPs) regulate synapse development by interacting with postsynaptic Slit- and Trk-like family proteins (Slitrks), which harbour two extracellular leucine-rich repeats (LRR1 and LRR2). Here we identify the minimal regions of the LAR-RPTPs and Slitrks, LAR-RPTPs Ig1–3 and Slitrks LRR1, for their interaction and synaptogenic function. Subsequent crystallographic and structure-guided functional analyses reveal that the splicing inserts in LAR-RPTPs are key molecular determinants for Slitrk binding and synapse formation. Moreover, structural comparison of the two Slitrk1 LRRs reveal that unique properties on the concave surface of Slitrk1 LRR1 render its specific binding to LAR-RPTPs. Finally, we demonstrate that lateral interactions between adjacent trans-synaptic LAR-RPTPs/Slitrks complexes observed in crystal lattices are critical for Slitrk1-induced lateral assembly and synaptogenic activity. Thus, we propose a model in which Slitrks mediate synaptogenic functions through direct binding to LAR-RPTPs and the subsequent lateral assembly of LAR-RPTPs/Slitrks complexes.

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Novel Glycosylated VEGF Decoy Receptor Fusion Protein, VEGF-Grab, Efficiently Suppresses Tumor Angiogenesis and Progression.
Novel Glycosylated VEGF Decoy Receptor Fusion Protein, VEGF-Grab, Efficiently Suppresses Tumor Angiogenesis and Progression.

    Anti-angiogenic therapies targeting vascular endothelial growth factor A (VEGFA) have been commonly used in clinics to treat cancers over the past decade. However, its clinical efficacy has been limited, with drawbacks including acquisition of resistance and activation of compensatory pathways resulting from elevated circulating VEGFB and placenta growth factor (PlGF). To bypass these disadvantages, we developed a novel glycosylated soluble decoy receptor fusion protein, VEGF-Grab, which can neutralize VEGFA, VEGFB, and PlGF. VEGF-Grab has the second and third immunoglobulin (Ig)-like domains of VEGF receptor 1 (VEGFR1) fused to IgG1 Fc, with three potential glycosylation sites introduced into the third Ig-like domain of VEGF-Grab by mutagenesis. Compared to VEGF-Trap, VEGF-Grab showed more potent decoy activity against VEGF and PlGF, mainly attributed to the VEGFR1 backbone. Most importantly, the negatively charged O-glycans attached to the third Ig-like domain of VEGFR1 counterbalanced the originally positively charged VEGFR1 backbone, minimizing non-specific binding of VEGF-Grab to the extracellular matrix, and resulting in greatly improved pharmacokinetic profile. These advancements led to stronger and more durable anti-angiogenic, anti-tumor, and anti-metastatic efficacy in both implanted and spontaneous tumor models as compared to VEGF-Trap, while toxicity profiles were comparable to VEGF-Trap. Collectively, our results highlight VEGF-Grab as a promising therapeutic candidate for further clinical drug development.

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Recombinant Lipase Engineered with Amphipathic and Coiled-Coil Peptides.
Recombinant Lipase Engineered with Amphipathic and Coiled-Coil Peptides.

    Lipases have been utilized industrially to produce biodiesel, oleochemicals, and pharmaceuticals. Many efforts such as metagenomics, directed evolution, and enzyme immobilization have been devoted to enhance the lipase activity. Here, we designed a recombinant lipase, NKC-M37-MAT, that was generated by incorporating an N-terminal amphipathic peptide (NKC) and a C-terminal coiled-coil peptide (MAT) into Photobacterium lipolyticum M37 lipase. The hydrophobic face of NKC improve the accessibility (Km), and catalytic efficiency (Kcat/Km) of the soluble lipase toward the hydrophobic substrate and tetrameric MAT further enhanced lipase catalytic activity (U/mg) through cooperative binding to its substrate such that the catalytic activity (U/mg) of NKC-M37-MAT was increased by a maximum of 54-fold compared with the wild-type, which decreased the biodiesel production time 5-fold from 30 to 6 h. This novel approach shows promise as a platform technology to increase lipase catalytic efficiency for industrial-scale production of biodiesel and biochemicals synthesized from hydrophobic substrates.

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A Designed Angiopoietin-1 Variant, Dimeric CMP-Ang1 Activates Tie2 and Stimulates Angiogenesis and Vascular Stabilization in N-glycan Dependent Manner.
A Designed Angiopoietin-1 Variant, Dimeric CMP-Ang1 Activates Tie2 and Stimulates Angiogenesis and Vascular Stabilization in N-glycan Dependent Manner.

    Angiopoietin-1 (Ang1), a potential growth factor for therapeutic angiogenesis and vascular stabilization, is known to specifically cluster and activateTie2 in high oligomeric forms, which is a unique and essential process in this ligand-receptor interaction. However, highly oligomeric native Ang1 and Ang1 variants are difficult to produce, purify, and store in a stable and active form. To overcome these limitations, we developed a simple and activedimeric CMP-Ang1 by replacing the N-terminal of native Ang1 with the coiled-coil domain of cartilage matrix protein (CMP) bearing mutations in its cysteine residues. This dimeric CMP-Ang1 effectively increased the migration, survival, and tube formation of endothelial cells via Tie2 activation. Furthermore, dimeric CMP-Ang1 induced angiogenesis and suppressed vascular leakage in vivo. Despite its dimeric structure, the potencies of suchTie2-activation-induced effects were comparable to those of a previously engineered protein, COMP-Ang1. We also revealed that these effects ofdimeric CMP-Ang1 were affected by specified N-glycosylation in its fibrinogen-like domain. Taken together, our results indicate that dimeric CMP-Ang1 is capable of activating Tie2 and stimulating angiogenesis in N-glycan dependent manner.

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Reconstruction of LPS Transfer Cascade Reveals Structural Determinants within LBP, CD14, and TLR4-MD2 for Efficient LPS Recognition and Transfer.
Reconstruction of LPS Transfer Cascade Reveals Structural Determinants within LBP, CD14, and TLR4-MD2 for Efficient LPS Recognition and Transfer.

    Lipopolysaccharide (LPS), the major component of the outer membrane of Gram-negative bacteria, binds Toll-like receptor 4 (TLR4)-MD2 complex and activates innate immune responses. LPS transfer to TLR4-MD2 is catalyzed by both LPS binding protein (LBP) and CD14. To define the sequential molecular interactions underlying this transfer, we reconstituted in vitro the entire LPS transfer process from LPS micelles to TLR4-MD2. Using electron microscopy and single-molecule approaches, we characterized the dynamic intermediate complexes for LPS transfer: LBP-LPS micelles, CD14-LBP-LPS micelle, and CD14-LPS-TLR4-MD2 complex. A single LBP molecule bound longitudinally to LPS micelles catalyzed multi-rounds of LPS transfer to CD14s that rapidly dissociated from LPB-LPS complex upon LPS transfer via electrostatic interactions. Subsequently, the single LPS molecule bound to CD14 was transferred to TLR4-MD2 in a TLR4-dependent manner. The definition of the structural determinants of the LPS transfer cascade to TLR4 may enable the development of targeted therapeutics for intervention in LPS-induced sepsis.

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Structural Insights into Modulation of Neurexin-Neuroligin Trans-synaptic Adhesion by MDGA1/Neuroligin-2 Complex.
Structural Insights into Modulation of Neurexin-Neuroligin Trans-synaptic Adhesion by MDGA1/Neuroligin-2 Complex.

    Membrane-associated mucin domain-containing glycosylphosphatidylinositol anchor proteins (MDGAs) bind directly to neuroligin-1 (NL1) and neuroligin-2 (NL2), thereby respectively regulating excitatory and inhibitory synapse development. However, the mechanisms by which MDGAs modulate NL activity to specify development of the two synapse types remain unclear. Here, we determined the crystal structures of human NL2/MDGA1 Ig1-3 complex, revealing their stable 2:2 arrangement with three interaction interfaces. Cell-based assays using structure-guided, site-directed MDGA1 mutants showed that all three contact patches were required for the MDGA’s negative regulation of NL2-mediated synaptogenic activity. Furthermore, MDGA1 competed with neurexins for NL2 via its Ig1 domain. The binding affinities of both MDGA1 and MDGA2 for NL1 and NL2 were similar, consistent with the structural prediction of similar binding interfaces. However, MDGA1 selectively associated with NL2, but not NL1, in vivo. These findings collectively provide structural insights into the mechanism by which MDGAs negatively modulate synapse development governed by NLs/neurexins.

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LAR-RPTP Clustering Is Modulated by Competitive Binding between Synaptic Adhesion Partners and Heparan Sulfate.
LAR-RPTP Clustering Is Modulated by Competitive Binding between Synaptic Adhesion Partners and Heparan Sulfate.

    The leukocyte common antigen-related receptor protein tyrosine phosphatases (LAR-RPTPs) are cellular receptors of heparan sulfate (HS) and chondroitin sulfate (CS) proteoglycans that direct axonal growth and neuronal regeneration. LAR-RPTPs are also synaptic adhesionmolecules that form trans-synaptic adhesion complexes by binding to various postsynaptic adhesion ligands, such as Slit- and Trk-like family of proteins (Slitrks), IL-1 receptor accessory protein-like 1 (IL1RAPL1), interleukin-1 receptor accessory protein (IL-1RAcP) and neurotrophin receptor tyrosine kinase C (TrkC), to regulate synaptogenesis. Here, we determined the crystal structure of the human LAR-RPTP/IL1RAPL1 complex and found that lateral interactions between neighboring LAR-RPTP/IL1RAPL1 complexes in crystal lattices are critical for the higher-order assembly and synaptogenic activity of these complexes. Moreover, we found that LAR-RPTP binding to the postsynaptic adhesionligands, Slitrk3, IL1RAPL1 and IL-1RAcP, but not TrkC, induces reciprocal higher-order clustering of trans-synaptic adhesion complexes. Although LAR-RPTP clustering was induced by either HS or postsynaptic adhesion ligands, the dominant binding of HS to the LAR-RPTPwas capable of dismantling pre-established LAR-RPTP-mediated trans-synaptic adhesion complexes. These findings collectively suggest that LAR-RPTP clustering for synaptogenesis is modulated by a complex synapse-organizing protein network.

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Four-fold Channel-Nicked Human Ferritin Nanocages for Active Drug Loading and pH-Responsive Drug Release.
Four-fold Channel-Nicked Human Ferritin Nanocages for Active Drug Loading and pH-Responsive Drug Release.

    Human ferritins are emerging platforms for non-toxic protein-based drug delivery, owing to their intrinsic or acquirable targeting abilities to cancer cells and hollow cage structures for drug loading. However, reliable strategies for high-level drug encapsulation within ferritin cavities and prompt cellular drug release are still lacking. Ferritin nanocages were developed with partially opened hydrophobic channels, which provide stable routes for spontaneous and highly accumulated loading of FeII -conjugated drugs as well as pH-responsive rapid drug releaseat endoplasmic pH. Multiple cancer-related compounds, such as doxorubicin, curcumin, and quercetin, were actively and heavily loaded onto the prepared nicked ferritin. Drugs on these minimally modified ferritins were effectively delivered inside cancer cells with high toxicity.

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Multi-paratopic VEGF decoy receptor have superior anti-tumor effects through anti-EGFRs and targeted anti-angiogenic activities.
Multi-paratopic VEGF decoy receptor have superior anti-tumor effects through anti-EGFRs and targeted anti-angiogenic activities.

    Limitation of current anti-Vascular Endothelial Growth Factor (VEGF) cancer therapy is transitory responses, inevitable relapses and its insufficient tumor-targeting. Thus, multifaceted approaches, including the development of bispecific antibodies and combination strategies targeting different pathways have been proposed as an alternative. Here, we developed a novel multi-paratopic VEGFdecoy receptor, Cetuximab-VEGF-Grab and Trastuzumab-VEGF-Grab, by genetically fusing VEGF decoy receptor (VEGF-Grab) to a single chain Fv of anti-Epidermal Growth Factor Receptor (EGFR) antibody (Cetuximab and Trastuzumab). These multi-paratopicVEGF decoy receptor, which recognize VEGF and EGFR family (EGFR or HER2), effectively suppressed both VEGF and EGFR pathways in vitro, to levels similar to those of the parental VEGF-Grab and anti-EGFR antibodies. In addition, the concurrent binding of multi-paratopic VEGF decoy receptor to VEGF and EGFR family enabled their specific localization to EGFR + tumor in vitro and in vivo. Furthermore, Cetuximab-VEGF-Grab and Trastuzumab-VEGF-Grab exhibited the enhanced anti-tumor activities compared to VEGF-Grab in EGFR + tumor xenograft mouse model via anti-EGFR and the targeted anti-angiogenic activities. These results indicate that multi-paratopic VEGF decoy receptor can be a promising agent, combining tumor-targeted anti-angiogenic therapy with efficient blockade of proliferative signals mediated by EGFR family.

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Publication

Pro-angiogenic Ginsenosides F1 and Rh1 Inhibit Vascular Leakage by Modulating NR4A1 Kang JI, Choi Y, Cui CH, Lee D, Kim SC, Kim HM Scientific Reports 2019; 9: 4502. 2019

Synaptic organizer: Slitrks and type IIa receptor protein tyrosine phosphatasess Seoung YounWon, PedroLee, Ho MinKim Current Opinion in Structural Biology 2019,54:95–103 2019

Insight Into the Interaction Between RNA Polymerase and VPg for Murine Norovirus Replication Lee J-H, Park BS, Han KR, Biering SB, Kim SJ, Choi J, Seok JH, Alam I, Chung MS, Kim HM, Hwang S, Kim KH frontiers in Microbiolo...

Multi-paratopic VEGF decoy receptor have superior anti-tumor effects through anti-EGFRs and target... Lee DH, Lee MY, Seo Y, Hong HJ, An HJ, Kang JS*, Kim HM* Biomaterials 2018 Apr 16;171:34-45 2018

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DMBL Disease Molecule Biochemistry Lab

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