Kim Myung-HeeUniversity of Kentucky, 800 Rose street,Dept of Biochemistry, Lexington, KY 40576-0084 USAPhone : 1 606 323 6629 Fax : 1 606323 1037 Send E-Mail to Kim Myung-Hee
Caffeoyl shikimate esterase (CSE) has been shown to play an important role in lignin biosynthesis in plants and is, therefore, a promising target for generating improved lignocellulosic biomass crops for sustainable biofuel production. Populus spp. has two CSE genes (CSE1 and CSE2) and, thus, the hybrid poplar (Populus alba x P. glandulosa) investigated in this study has four CSE genes. Here, we present transgenic hybrid poplars with knockouts of each CSE gene achieved by CRISPR/Cas9. To knockout the CSE genes of the hybrid poplar, we designed three single guide RNAs (sg1-sg3), and produced three different transgenic poplars with either CSE1 (CSE1-sg2), CSE2 (CSE2-sg3), or both genes (CSE1/2-sg1) mutated. CSE1-sg2 and CSE2-sg3 poplars showed up to 29.1% reduction in lignin deposition with irregularly shaped xylem vessels. However, CSE1-sg2 and CSE2-sg3 poplars were morphologically indistinguishable from WT and showed no significant differences in growth in a long-term living modified organism (LMO) field-test covering four seasons. Gene expression analysis revealed that many lignin biosynthetic genes were downregulated in CSE1-sg2 and CSE2-sg3 poplars. Indeed, the CSE1-sg2 and CSE2-sg3 poplars had up to 25% higher saccharification efficiency than the WT control. Our results demonstrate that precise editing of CSE by CRISPR/Cas9 technology can improve lignocellulosic biomass without a growth penalty.
Title: Superior immune responses induced by intranasal immunization with recombinant adenovirus-based vaccine expressing full-length Spike protein of Middle East respiratory syndrome coronavirus Kim MH, Kim HJ, Chang J Ref: PLoS ONE, 14:e0220196, 2019 : PubMed
Middle East respiratory syndrome coronavirus (MERS-CoV) causes an acute and severe lower respiratory illness as well as vomiting, diarrhea, and renal failure. Because no licensed MERS-CoV vaccines are currently available, preventive and therapeutic measures are urgently needed. The surface spike (S) glycoprotein of MERS-CoV, which binds to the cellular receptor dipeptidyl peptidase 4 (DPP4), is considered as a major target for MERS-CoV vaccine development. Here, we designed recombinant replication-deficient adenovirus-based vaccines expressing the N-terminal domain (rAd/NTD) and receptor-binding domain (rAd/RBD) of the MERS-CoV S1 subunit and full-length Spike protein (rAd/Spike). We found that immunization with candidate vaccines via intranasal route induced S1-specific IgG antibodies and neutralizing antibodies against MERS spike pseudotyped virus. Especially, rAd/Spike induced the highest neutralizing antibody titer and the strongest cytokine-induced T cell responses among the three candidate vaccines. To compare the immune responses induced by different administration routes, rAd/Spike was administered via intranasal, sublingual, or intramuscular route. All these administration routes exhibited neutralizing effects in the serum. MERS-CoV-specific neutralizing IgA antibodies in the bronchoalveolar lavage fluid were only induced by intranasal and sublingual administration but not by intramuscular administration. Intranasal administration with rAd/Spike also created resident memory CD8 T cells in the airway and lung parenchyma. Taken together, our results showed that both the humoral and cellular immune responses are highly induced by rAd/Spike administration, suggesting that rAd/Spike may confer protection against MERS-CoV infection.
Upon invading target cells, multifunctional autoprocessing repeats-in-toxin (MARTX) toxins secreted by bacterial pathogens release their disease-related modularly structured effector domains. However, it is unclear how a diverse repertoire of effector domains within these toxins are processed and activated. Here, we report that Makes caterpillars floppy-like effector (MCF)-containing MARTX toxins require ubiquitous ADP-ribosylation factor (ARF) proteins for processing and activation of intermediate effector modules, which localize in different subcellular compartments following limited processing of holo effector modules by the internal cysteine protease. Effector domains structured tandemly with MCF in intermediate modules become disengaged and fully activated by MCF, which aggressively interacts with ARF proteins present at the same location as intermediate modules and is converted allosterically into a catalytically competent protease. MCF-mediated effector processing leads ultimately to severe virulence in mice via an MCF-mediated ARF switching mechanism across subcellular compartments. This work provides insight into how bacteria take advantage of host systems to induce systemic pathogenicity.
In this study, pharmacophore based 3D QSAR models for human acetylcholinesterase (AChE) inhibitors were generated, with good significance, statistical values (r(2)training = 0.73) and predictability (q(2)training = 0.67). It was further validated by three methods (Fischer's test, decoy set and Guner-Henry scoring method) to show that the models can be used to predict the biological activities of compounds without costly and time-consuming synthesis. The criteria for virtual screening were also validated by testing the selective AChE inhibitors. Virtual screening experiments and subsequent in vitro evaluation of promising hits revealed a novel and selective AChE inhibitor. Thus, the findings reported herein may provide a new strategy for the discovery of selective AChE inhibitors. The IC50 value of compounds 5c and 6a presented selective inhibition of AChE without inhibiting butyrylcholinesterase (BChE) at uM level. Molecular docking studies were performed to explain the potent AChE inhibition of the target compounds studies to explain high affinity.
PURPOSE: Plasma lipoprotein-associated phospholipase A (Lp-PLA) binds to low-density lipoprotein. The levels of Lp-PLA reflect the plaque burden, and are upregulated in acute coronary syndrome (ACS). We investigated the diagnostic value of Lp-PLA2 levels and found that it might be a potential biomarker for ACS. MATERIALS AND METHODS: We classified 226 study participants into three groups: patients without significant stenosis (control group), patients with significant stenosis with stable angina (SA group), and patients with ACS (ACS group). RESULTS: Lp-PLA and high-sensitivity C-reactive protein (hs-CRP) levels were significantly greater in the ACS group than in the SA group (p=0.044 and p=0.029, respectively). Multivariate logistic regression analysis revealed that Lp-PLA levels are significantly associated with ACS (odds ratio=1.047, p=0.013). The addition of Lp-PLA to the ACS model significantly increased the global ^2 value over traditional risk factors (28.14 to 35.602, p=0.006). The area under the receiver operating characteristic curve for Lp-PLA was 0.624 (p=0.004). The addition of Lp-PLA level to serum hs-CRP concentration yielded an integrated discrimination improvement of 0.0368 (p=0.0093, standard error: 0.0142) and improved the ability to diagnose ACS. CONCLUSION: Lp-PLA levels are related to plaque stability and might be a diagnostic biomarker for ACS.
Synaptic adhesion molecules regulate diverse aspects of synapse formation and maintenance. Many known synaptic adhesion molecules localize at excitatory synapses, whereas relatively little is known about inhibitory synaptic adhesion molecules. Here we report that IgSF9b is a novel, brain-specific, homophilic adhesion molecule that is strongly expressed in GABAergic interneurons. IgSF9b was preferentially localized at inhibitory synapses in cultured rat hippocampal and cortical interneurons and was required for the development of inhibitory synapses onto interneurons. IgSF9b formed a subsynaptic domain distinct from the GABAA receptor- and gephyrin-containing domain, as indicated by super-resolution imaging. IgSF9b was linked to neuroligin 2, an inhibitory synaptic adhesion molecule coupled to gephyrin, via the multi-PDZ protein S-SCAM. IgSF9b and neuroligin 2 could reciprocally cluster each other. These results suggest a novel mode of inhibitory synaptic organization in which two subsynaptic domains, one containing IgSF9b for synaptic adhesion and the other containing gephyrin and GABAA receptors for synaptic transmission, are interconnected through S-SCAM and neuroligin 2.
Considerable attention has recently been paid to the N-Myc downstream-regulated gene (NDRG) family because of its potential as a tumor suppressor in many human cancers. Primary amino acid sequence information suggests that the NDRG family proteins may belong to the alpha/beta-hydrolase (ABH) superfamily; however, their functional role has not yet been determined. Here, we present the crystal structures of the human and mouse NDRG2 proteins determined at 2.0 and 1.7 A resolution, respectively. Both NDRG2 proteins show remarkable structural similarity to the ABH superfamily, despite limited sequence similarity. Structural analysis suggests that NDRG2 is a nonenzymatic member of the ABH superfamily, because it lacks the catalytic signature residues and has an occluded substrate-binding site. Several conserved structural features suggest NDRG may be involved in molecular interactions. Mutagenesis data based on the structural analysis support a crucial role for helix alpha6 in the suppression of TCF/beta-catenin signaling in the tumorigenesis of human colorectal cancer, via a molecular interaction.
MELDB is a comprehensive protein database of microbial esterases and lipases which are hydrolytic enzymes important in the modern industry. Proteins in MELDB are clustered into groups according to their sequence similarities based on a local pairwise alignment algorithm and a graph clustering algorithm (TribeMCL). This differs from traditional approaches that use global pairwise alignment and joining methods. Our procedure was able to reduce the noise caused by dubious alignment in the distantly related or unrelated regions in the sequences. In the database, 883 esterase and lipase sequences derived from microbial sources are deposited and conserved parts of each protein are identified. HMM profiles of each cluster were generated to classify unknown sequences. Contents of the database can be keyword-searched and query sequences can be aligned to sequence profiles and sequences themselves.
Title: New cold-adapted lipase from Photobacterium lipolyticum sp. nov. that is closely related to filamentous fungal lipases Ryu HS, Kim HK, Choi WC, Kim MH, Park SY, Han NS, Oh TK, Lee JK Ref: Applied Microbiology & Biotechnology, 70:321, 2006 : PubMed
A Photobacterium strain, M37, showing lipolytic activity, was previously isolated from an intertidal flat of the Yellow Sea in Korea and identified as Photobacterium lipolyticum sp. nov. In the present study, the corresponding gene was cloned using the shotgun method. The amino acid sequence deduced from the nucleotide sequence (1,023 bp) corresponded to a protein of 340 amino acid residues with a molecular weight of 38,026. No sequence similarity was found with any known bacterial lipases/esterases; instead, the most similar enzymes were several filamentous fungal lipases. Although the similarity was very low (less than 16%), there were many conserved regions over the entire sequence and N-terminal oxyanion hole (RG) region, a signature sequence of filamentous fungal lipases. The novel protein M37 was produced in both a soluble and insoluble form when the Escherichia coli cells harboring the gene were cultured at 18 degrees C. The soluble protein exhibited lipase activity in a pH-stat assay using an olive oil emulsion. The M37 lipase also displayed a maximum activity at 25 degrees C and maintained its activity at a low temperature range (5-25 degrees C) with an activation energy (E(a)) of 2.07 kcal/mol. Accordingly, these results indicate that the M37 lipase from P. lipolyticum sp. nov. is a new cold-adapted enzyme.
Title: Zinc in lipase L1 from Geobacillus stearothermophilus L1 and structural implications on thermal stability Choi WC, Kim MH, Ro HS, Ryu SR, Oh TK, Lee JK Ref: FEBS Letters, 579:3461, 2005 : PubMed
Lipase L1 from Geobacillus stearothermophilus L1 contains an unusual extra domain, making a tight intramolecular interaction with the main catalytic domain through a Zn2+-binding coordination. To elucidate the role of the Zn2+, we disrupted the Zn2+-binding site by mutating the zinc-ligand residues (H87A, D61A/H87A, and D61A/H81A/H87A/D238A). The activity vs. temperature profiles of the mutant enzymes showed that the disruption of the Zn2+-binding site resulted in a notable decrease in the optimal temperature for maximal activity from 60 to 45-50 degrees C. The mutations also abolished the Zn2+-induced thermal stabilization. The wild-type enzyme revealed a 34.6-fold increase in stabilization with the addition of Zn2+ at 60 degrees C, whereas the mutant enzymes exhibited no response to Zn2+. Additional circular dichroism spectroscopy studies also confirmed the structural stabilizing role of Zn2+ on lipase L1 at elevated temperatures.
Title: The vesicular acetylcholine transporter interacts with clathrin-associated adaptor complexes AP-1 and AP-2 Kim MH, Hersh LB Ref: Journal of Biological Chemistry, 279:12580, 2004 : PubMed
In neuronal cells the neurotransmitter acetylcholine is transferred from the cytoplasm into synaptic vesicles by the vesicular acetylcholine transporter (VAChT). The cytoplasmic tail of VAChT has been shown to contain signals that direct its sorting and trafficking. The role of clathrin-associated protein complexes in VAChT sorting to synaptic vesicles has been examined. A fusion protein between the VAChT cytoplasmic tail and glutathione S-transferase was used to identify VAChT-clathrin-associated protein adaptor protein 1, adaptor protein 2 and adaptor protein 180 complexes from a rat brain extract. In vivo coimmunoprecipitation confirmed adaptin alpha and adaptin gamma complexes, but adaptor protein 180 complexes were not detected by this technique. Deletion and site directed mutagenesis show that the VAChT cytoplasmic tail contains multiple trafficking signals. These include a non-classical tyrosine motif that serves as the signal for adaptin alpha and a dileucine motif that serves as the signal for adaptin gamma. A classical tyrosine motif is also involved in VAChT trafficking, but does not interact with any known adaptor proteins. There appear to be two endocytosis motifs, one involving the adaptor protein 1 binding site and the other involving the adaptor protein 2 binding site. These results suggest a complex trafficking pathway for VAChT.
The protein kinase A-deficient PC12 cell line PC12A123.7 lacks both choline acetyltransferase and the vesicular acetylcholine transporter. This cell line has been used to establish a stably transfected cell line expressing recombinant rat vesicular acetylcholine transporter that is appropriately trafficked to small synaptic vesicles. Acetylcholine is transported by the rat vesicular acetylcholine transporter at a maximal rate of 1.45 nmol acetylcholine/min/mg protein and exhibits a Km for transport of 2.5 mM. The transporter binds vesamicol with a Kd of 7.5 nM. The ability of structural analogs of acetylcholine to inhibit both acetylcholine uptake and vesamicol binding was measured. The results demonstrate that like Torpedo vesicular acetylcholine transporter, the mammalian transporter can bind a diverse group of acetylcholine analogs.
Title: Expression and characterization of Ca(2+)-independent lipase from Bacillus pumilus B26 Kim HK, Choi HJ, Kim MH, Sohn CB, Oh TK Ref: Biochimica & Biophysica Acta, 1583:205, 2002 : PubMed
A lipase-producing Bacillus pumilus strain (B26) was isolated from a soil sample collected in Korea. The cloned gene showed that the lipase B26 composed of a 34-amino-acid signal sequence and a 181-amino-acid mature part corresponding to a molecular mass (M(r)) of 19,225. Based on the M(r) and the protein sequence, the lipase B26 belongs to the lipase family I.4. The optimum temperature and pH of the purified enzyme were 35 degrees C and 8.5, respectively. The lipase B26 showed a 'Ca(2+)-independent thermostability and catalytic activity'. These are novel properties observed for the first time in lipase B26 among all bacterial lipases and correspond with the suggestion that this enzyme had no Ca(2+)-binding motif around the catalytic His156 residue. This enzyme seems to be a true lipase based on the experimental results that it could hydrolyze various long-chain triglycerides (C(14)-C(18)) and triolein (C(18:1)) and that it showed a typical interfacial activation mechanism toward both tripropionin and p-nitrophenyl butyrate.
Metabolic labeling of a mutant PC12 cell line, A123.7, expressing recombinant rat vesicular acetylcholine transporter (VAChT) with radiolabeled inorganic phosphate was used to demonstrate phosphorylation of the transporter on a serine residue. Mutational analysis was used to demonstrate that serine 480, which is located on the COOH-terminal cytoplasmic tail, is the sole phosphorylation site. Phosphorylation of serine 480 was attributable to the action of protein kinase C. Using a permanently dephosphorylated form of rat VAChT, S480A rVAChT, it was shown that this mutant displays the same kinetics for the transport of acetylcholine and the binding of the inhibitor vesamicol as does the wild type transporter. However, sucrose gradient density centrifugation showed that, unlike wild type VAChT, the S480A mutant did not localize to synaptic vesicles. These results suggest that phosphorylation of serine 480 of VAChT is involved in the trafficking of this transporter.
Title: Mutational analysis of basic residues in the rat vesicular acetylcholine transporter. Identification of a transmembrane ion pair and evidence that histidine is not involved in proton translocation Kim MH, Lu M, Kelly M, Hersh LB Ref: Journal of Biological Chemistry, 275:6175, 2000 : PubMed
The function of positively charged residues and the interaction of positively and negatively charged residues of the rat vesicular acetylcholine transporter (rVAChT) were studied. Changing Lys-131 in transmembrane domain helix 2 (TM2) to Ala or Leu eliminated transport activity, with no effect on vesamicol binding. However, replacement by His or Arg retained transport activity, suggesting a positive charge in this position is critical. Mutation of His-444 in TM12 or His-413 in the cytoplasmic loop between TM10 and TM11 was without effect on ACh transport, but vesamicol binding was reduced with His-413 mutants. Changing His-338 in TM8 to Ala or Lys did not effect ACh transport, however replacement with Cys or Arg abolished activity. Mutation of both of the transmembrane histidines or all three of the luminal loop histidines showed no change in acetylcholine transport. The mutant H338A/D398N between oppositely charged residues in transmembrane domains showed no vesamicol binding, however the charge reversal mutant H338D/D398H restored binding. This suggests that His-338 forms an ion pair with Asp-398. The charge neutralizing mutant K131A/D425N or the charge exchanged mutant K131D/D425K did not restore ACh transport. Taken together these results provide new insights into the tertiary structure in VAChT.
Title: Mutational analysis of aspartate residues in the transmembrane regions and cytoplasmic loops of rat vesicular acetylcholine transporter Kim MH, Lu M, Lim EJ, Chai YG, Hersh LB Ref: Journal of Biological Chemistry, 274:673, 1999 : PubMed
The vesicular acetylcholine transporter (VAChT) is responsible for the transport of the neurotransmitter acetylcholine (ACh) into synaptic vesicles using an electrochemical gradient to drive transport. Rat VAChT has a number of aspartate residues within its predicted transmembrane domains (TM) and cytoplasmic loops, which may play important structural or functional roles in acetylcholine transport. In order to identify functional charged residues, site-directed mutagenesis of rVAChT was undertaken. No effect on ACh transport was observed when any of the five aspartate residues in the cytoplasmic loop were converted to asparagine. Similarly, changing Asp-46 (D46N) in TM1 or Asp-255 (D255N) in TM6 had no effect on ACh transport or vesamicol binding. However, replacement of Asp-398 in TM10 with Asn completely eliminated both ACh transport and vesamicol binding. The conservative mutant D398E retained transport activity, but not vesamicol binding, suggesting this residue is critical for transport. Mutation of Asp-193 in TM4 did not affect ACh transport activity; however, vesamicol binding was dramatically reduced. With mutant D425N of TM11 transport activity for ACh was completely blocked, without an effect on vesamicol binding. Activity was not restored in the conservative mutant D425E, suggesting the side chain as well as the negative charge of Asp-425 is important for substrate binding. These mutants, as well as mutant D193N, clearly dissociated ACh binding and transport from vesamicol binding. These data suggest that Asp-398 in TM10 and Asp-425 in TM11 are important for ACh binding and transport, while Asp-193 and Asp-398 in TM4 and TM10, respectively, are involved in vesamicol binding.
Title: The role of charged transmembrane residues of rVAChT on ACh transport and vesamicol binding Kim MH, Lu M, Lim EJ, Chai YG, Hersh LB Ref: Journal de Physiologie (Paris), 92:448, 1998 : PubMed
