Kuglae Kim

Akt-mediated phosphorylation increases the binding affinity of hTERT for importin α to promote nuclear translocation.

ABSTRACT Telomeres are essential for chromosome integrity and protection, and their maintenance requires the ribonucleoprotein enzyme telomerase. Previously, we have shown that human telomerase reverse transcriptase (hTERT) contains a bipartite nuclear localization signal (NLS; residues 222–240) that is responsible for nuclear import, and that Akt-mediated phosphorylation of residue S227 is important for efficient nuclear import of hTERT. Here, we show that hTERT binds to importin-α proteins through the bipartite NLS and that this heterodimer then forms a complex with importin-β proteins to interact with the nuclear pore complex. Depletion of individual importin-α proteins results in a failure of hTERT nuclear import, and the resulting cytoplasmic hTERT is degraded by ubiquitin-dependent proteolysis. Crystallographic analysis reveals that the bipartite NLS interacts with both the major and minor sites of importin-α proteins. We also show that Akt-mediated phosphorylation of S227 increases the binding affinity for importin-α proteins and promotes nuclear import of hTERT, thereby resulting in increased telomerase activity. These data provide details of a binding mechanism that enables hTERT to interact with the nuclear import receptors and of the control of the dynamic nuclear transport of hTERT through phosphorylation. Summary: Akt-mediated phosphorylation of residue S227 within the nuclear localization signal of hTERT increases the binding affinity for importin α and promotes nuclear import of hTERT.

Structural Basis for the Inhibition of Human Lysozyme by PliC from Brucella abortus

Lysozymes are the first line of defense for a diverse range of organisms that catalyze the degradation of bacterial peptidoglycan. Gram-negative bacteria produce proteinaceous lysozyme inhibitors to protect themselves from the action of lysozymes. To date, MliC or PliC (membrane-bound or periplasmic inhibitor of c-type lysozyme, respectively) has been found in various Gram-negative bacteria. Here, we report the crystal structures of Brucella abortus PliC and its complex with human c-type lysozyme. The complex structure demonstrates that the invariant loop of MliC/PliC plays a crucial role in the inhibition of lysozyme via its insertion into the active site cleft of the lysozyme, as previously observed in the complex structure of Pseudomonas aeruginosa MliC and chicken c-type lysozyme. We identified a new binding interface between a loop adjacent to the active site of human lysozyme and a loop carrying Glu112 of B. abortus PliC, the structure of which was disordered in P. aeruginosa MliC. Because MliC/PliC family members have been implicated as putative colonization or virulence factors, the structures and mechanism of action of MliC/PliC will be relevant to the control of bacterial growth in animal hosts.

Monomerization and ER Relocalization of GRASP Is a Requisite for Unconventional Secretion of CFTR.

Induction of endoplasmic reticulum (ER)‐to‐Golgi blockade or ER stress induces Golgi reassembly stacking protein (GRASP)‐mediated, Golgi‐independent unconventional cell‐surface trafficking of the folding‐deficient ΔF508‐cystic fibrosis transmembrane conductance regulator (CFTR). However, molecular mechanisms underlying this process remain elusive. Here, we show that phosphorylation‐dependent dissociation of GRASP homotypic complexes and subsequent relocalization of GRASP to the ER play a critical role in the unconventional secretion of CFTR. Immunolocalization analyses of mammalian cells revealed that the Golgi protein GRASP55 was redistributed to the ER by stimuli that induce unconventional secretion of ΔF508‐CFTR, such as induction of ER‐to‐Golgi blockade by the Arf1 mutant. Notably, the same stimuli also induced phosphorylation of regions near the C‐terminus of GRASP55 and dissociation of GRASP homomultimer complexes. Furthermore, phosphorylation‐mimicking mutations of GRASP55 induced the monomerization and ER relocalization of GRASP55, and these changes were nullified by phosphorylation‐inhibiting mutations. These results provide mechanistic insights into how GRASP accesses the ER‐retained ΔF508‐CFTR and mediates the ER stress‐induced unconventional secretion pathway.

The Crystal Structure of Mpk38 in Complex with Otssp167, an Orally Administrative Melk Selective Inhibitor.

Murine protein serine/threonine kinase 38 (MPK38), also known as maternal embryonic leucine zipper kinase (MELK), has been associated with various human cancers and plays an important role in the formation of cancer stem cells. OTSSP167, a MELK selective inhibitor, exhibits a strong in vitro activity, conferring an IC50 of 0.41nM and in vivo effect on various human cancer xenograft models. Here, we report the crystal structure of MPK38 (T167E), an active mutant, in complex with OTSSP167 and describe its detailed protein-inhibitor interactions. Comparison with the previous determined structure of MELK bound to the nanomolar inhibitors shows that OTSSP167 effectively fits into the active site, thus offering an opportunity for structure-based development and optimization of MELK inhibitors.

Solution structure of the transmembrane 2 domain of the human melanocortin-4 receptor in sodium dodecyl sulfate (SDS) micelles and the functional implication of the D90N mutant

The melanocortin receptors (MCRs) are members of the G protein-coupled receptor (GPCR) 1 superfamily with seven transmembrane (TM) domains. Among them, the melanocortin-4 receptor (MC4R) subtype has been highlighted recently by genetic studies in obese humans. In particular, in a patient with severe early-onset obesity, a novel heterozygous mutation in the MC4R gene was found in an exchange of Asp to Asn in the 90th amino acid residue located in the TM 2 domain (MC4RD90N). Mutations in the MC4R gene are the most frequent monogenic causes of severe obesity and are described as heterozygous with loss of function. We determine solution structures of the TM 2 domain of MC4R (MC4RTM2) and compared secondary structure of Asp90 mutant (MC4RTM2-D90N) in a micelle environment by nuclear magnetic resonance (NMR) spectroscopy. NMR structure shows that MC4RTM2 forms a long α-helix with a kink at Gly98. Interestingly, the structure of MC4RTM2-D90N is similar to that of MC4RTM2 based on data from CD and NMR spectrum. However, the thermal stability and homogeneity of MC4RD90N is quite different from those of MC4R. The structure from molecular modeling suggests that Asp90(2.50) plays a key role in allosteric sodium ion binding. Our data suggest that the sodium ion interaction of Asp90(2.50) in the allosteric pocket of MC4R is essential to its function, explaining the loss of function of the MC4RD90N mutant.

GC1118, an anti-EGFR antibody with a distinct binding epitope and superior inhibitory activity against high-affinity EGFR ligands

The EGFR-targeted monoclonal antibodies are a valid therapeutic strategy for patients with metastatic colorectal cancer (mCRC). However, only a small subset of mCRC patients has therapeutic benefits and there are high demands for EGFR therapeutics with a broader patient pool and more potent efficacy. In this study, we report GC1118 exhibiting a different character in terms of binding epitope, affinity, mode of action, and efficacy from other anti-EGFR antibodies. Structural analysis of the EGFR–GC1118 crystal complex revealed that GC1118 recognizes linear, discrete N-terminal epitopes of domain III of EGFR, critical for EGF binding but not overlapping with those of other EGFR-targeted antibodies. GC1118 exhibited superior inhibitory activity against high-affinity EGFR ligands in terms of EGFR binding, triggering EGFR signaling, and proliferation compared with cetuximab and panitumumab. EGFR signaling driven by low-affinity ligands, on the contrary, was well inhibited by all the antibodies tested. GC1118 demonstrated robust antitumor activity in tumor xenografts with elevated expression of high-affinity ligands in vivo, whereas cetuximab did not. Considering the significant role of high-affinity EGFR ligands in modulating tumor microenvironment and inducing resistance to various cancer therapeutics, our study suggests a potential therapeutic advantage of GC1118 in terms of efficacy and a range of benefited patient pool. Mol Cancer Ther; 15(2); 251–63. ©2015 AACR.

In Vitro and In Vivo Investigation of the Inhibition of Trypanosoma brucei Cell Growth by Lipophilic Bisphosphonates

ABSTRACT We report the results of a screen of a library of 925 potential prenyl synthase inhibitors against Trypanosoma brucei farnesyl diphosphate synthase (TbFPPS) and against T. brucei, the causative agent of human African trypanosomiasis. The most potent compounds were lipophilic analogs of the bone resorption drug zoledronate, some of which had submicromolar to low micromolar activity against bloodstream form T. brucei and selectivity indices of up to ∼300. We evaluated the effects of two such inhibitors on survival and parasitemia in a T. brucei mouse model of infection and found that survival increased by up to 16 days. We also investigated the binding of three lipophilic bisphosphonates to an expressed TbFPPS using crystallography and investigated the thermodynamics of binding using isothermal titration calorimetry.

IgE cross-reactivity of peanut with walnut and soybean in children with food allergy.

BACKGROUND Peanut allergies are common and can be life-threating for sensitised individuals. Peanut allergens share significant amino acid homology with those of other legumes and tree nuts, but their cross-reactivity still remains unclear. OBJECTIVE We sought to determine the clinical significance of the cross-reactivity of peanut allergens with those of walnut and soybean. METHODS Pooled sera from eight subjects with both peanut and walnut specific IgE were investigated in an inhibition test. After the sera were incubated with either peanut or walnut protein extracts, the quantity of IgE antibodies against the peanut and walnut was measured using an immunoCAP test. Likewise, pooled sera from 18 subjects with both peanut and soybean specific IgE antibodies were incubated with either peanut or soybean protein extracts and evaluated with a peanut and soybean immunoCAP test. SDS-PAGE and immunoblotting were also performed with peanut, walnut and soybean protein extracts and relevant sera. RESULTS Peanut specific IgE was inhibited up to 20% and 26% by walnut and soybean protein extracts, respectively. In reverse, walnut and soybean specific IgE were inhibited up to 21% and 23% by peanut protein extracts, respectively. In the immunoblot analysis, pooled serum from the subjects with peanut specific IgE antibodies reacted with walnut protein extracts significantly. CONCLUSION Although the clinical significance of the cross-reactivity of peanut specific IgE with walnut and soybean protein extracts has not been established, we believe that individuals who are allergic to peanuts need to be cautious about consuming walnuts and soybeans.

Co-expression of human agouti-related protein enhances expression and stability of human melanocortin-4 receptor

G protein-coupled receptors (GPCRs) represent the largest family of transmembrane signaling proteins, and they are considered major targets of approximately half of all therapeutic agents. Human melanocortin-4 receptor (hMC4R) plays an important role in the control of energy homeostasis, and its mutants are directly related to severe human obesity. Here, we describe optimized protocols for the high-yield expression and purification of hMC4R that will accelerate structural study. Truncations of the N- and C-termini of hMC4R with T4 lysozyme (T4L) insertion increase the solubility as well as stability of the protein. Strikingly, co-expression of human mini-agouti-related protein (mini-AgRP) in Spodoptera frugiperda (Sf9) cells enables excellent stability of hMC4R. The protein yield in the human mini-AgRP co-expression system is increased by about 3-4 times compared to that of hMC4R alone. Data from analytical size exclusion chromatography (aSEC) and thermostability assay show that hMC4R becomes homogeneous and stable with a melting temperature of 58°C in the presence of human mini-AgRP.

Crystal Structure of Human Dual-Specificity Tyrosine-Regulated Kinase 3 Reveals New Structural Features and Insights into its Auto-phosphorylation

Dual-specificity tyrosine-regulated kinases (DYRKs) auto-phosphorylate a critical tyrosine residue in their activation loop and phosphorylate their substrate on serine and threonine residues. The auto-phosphorylation occurs intramolecularly and is a one-off event. DYRK3 is selectively expressed at a high level in hematopoietic cells and attenuates erythroblast development, leading to anemia. In the present study, we determined the crystal structure of the mature form of human DYRK3 in complex with harmine, an ATP competitive inhibitor. The crystal structure revealed a phosphorylation site, residue S350, whose phosphorylation increases the stability of DYRK3 and enhances its kinase activity. In addition, our structural and biochemical assays suggest that the N-terminal auto-phosphorylation accessory domain stabilizes the DYRK3 protein, followed by auto-phosphorylation of the tyrosine of the activation loop, which is important for kinase activity. Finally, our docking analysis provides information for the design of novel and potent therapeutics to treat anemia.