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Abstract HEPN–MNT, a type VII TA module, comprises the HEPN toxin and the MNT antitoxin, which acts as a nucleotidyltransferase that transfers the NMP moiety to the corresponding HEPN toxin, thereby interfering with its toxicity. Here, we report crystal structures of the Legionella pneumophila HEPN–MNT module, including HEPN, AMPylated HEPN, MNT, and the HEPN–MNT complex. Our structural analysis and biochemical assays, suggest that HEPN is a metal-dependent RNase and identify its active site residues. We also elucidate the oligomeric state of HEPN in solution. Interestingly, L. pneumophila MNT, which lacks a long C-terminal α4 helix, controls the toxicity of HEPN toxin via a distinct binding mode with HEPN. Finally, we propose a comprehensive regulatory mechanism of the L. pneumophila HEPN–MNT module based on structural and functional studies. These results provide insight into the type VII HEPN–MNT TA system.

Abstract Background Despite advancements in chronic myeloid leukemia (CML) therapy with tyrosine kinase inhibitors (TKIs), resistance and intolerance remain significant challenges. Leukemia stem cells (LSCs) and TKI-resistant cells rely on altered mitochondrial metabolism and oxidative phosphorylation. Targeting rewired energy metabolism and inducing non-apoptotic cell death, along with the release of damage-associated molecular patterns (DAMPs), can enhance therapeutic strategies and immunogenic therapies against CML and prevent the emergence of TKI-resistant cells and LSC persistence. Methods Transcriptomic analysis was conducted using datasets of CML patients' stem cells and healthy cells. DNA damage was evaluated by fluorescent microscopy and flow cytometry. Cell death was assessed by trypan blue exclusion test, fluorescent microscopy, flow cytometry, colony formation assay, and in vivo Zebrafish xenografts. Energy metabolism was determined by measuring NAD+ and NADH levels, ATP production rate by Seahorse analyzer, and intracellular ATP content. Mitochondrial fitness was estimated by measurements of mitochondrial membrane potential, ROS, and calcium accumulation by flow cytometry, and morphology was visualized by TEM. Bioinformatic analysis, real-time qPCR, western blotting, chemical reaction prediction, and molecular docking were utilized to identify the drug target. The immunogenic potential was assessed by high mobility group box (HMGB)1 ELISA assay, luciferase-based extracellular ATP assay, ectopic calreticulin expression by flow cytometry, and validated by phagocytosis assay, and in vivo vaccination assay using syngeneic C57BL/6 mice. Results Transcriptomic analysis identified metabolic alterations and DNA repair deficiency signatures in CML patients. CML patients exhibited enrichment in immune system, DNA repair, and metabolic pathways. The gene signature associated with BRCA mutated tumors was enriched in CML datasets, suggesting a deficiency in double-strand break repair pathways. Additionally, poly(ADP-ribose) polymerase (PARP)1 was significantly upregulated in CML patients’ stem cells compared to healthy counterparts. Consistent with the CML patient DNA repair signature, treatment with the methylated indolequinone MAC681 induced DNA damage, mitochondrial dysfunction, calcium homeostasis disruption, metabolic catastrophe, and necroptotic-like cell death. In parallel, MAC681 led to PARP1 degradation that was prevented by 3-aminobenzamide. MAC681-treated myeloid leukemia cells released DAMPs and demonstrated the potential to generate an immunogenic vaccine in C57BL/6 mice. MAC681 and asciminib exhibited synergistic effects in killing both imatinib-sensitive and -resistant CML, opening new therapeutic opportunities. Conclusions Overall, increasing the tumor mutational burden by PARP1 degradation and mitochondrial deregulation makes CML suitable for immunotherapy.

Abstract Malaria poses an enormous threat to human health. With ever increasing resistance to currently deployed drugs, breakthrough compounds with novel mechanisms of action are urgently needed. Here, we explore pyrimidine-based sulfonamides as a new low molecular weight inhibitor class with drug-like physical parameters and a synthetically accessible scaffold. We show that the exemplar, OSM-S-106, has potent activity against parasite cultures, low mammalian cell toxicity and low propensity for resistance development. In vitro evolution of resistance using a slow ramp-up approach pointed to the Plasmodium falciparum cytoplasmic asparaginyl-tRNA synthetase (PfAsnRS) as the target, consistent with our finding that OSM-S-106 inhibits protein translation and activates the amino acid starvation response. Targeted mass spectrometry confirms that OSM-S-106 is a pro-inhibitor and that inhibition of PfAsnRS occurs via enzyme-mediated production of an Asn-OSM-S-106 adduct. Human AsnRS is much less susceptible to this reaction hijacking mechanism. X-ray crystallographic studies of human AsnRS in complex with inhibitor adducts and docking of pro-inhibitors into a model of Asn-tRNA-bound PfAsnRS provide insights into the structure-activity relationship and the selectivity mechanism.

Objective: Alterations in lipid metabolism are associated with aging and age-related diseases. Chaperone-mediated autophagy (CMA) is a lysosome-dependent process involved in specific protein degradation. Heat shock cognate 71 kDa protein (Hsc70) recognizes cytosolic proteins with KFERQ motif and allows them to enter the lysosome via lysosome-associated membrane glycoprotein 2 isoform A (LAMP2A). CMA deficiency is associated with dysregulated lipid metabolism in the liver. In this study, we examined the effect of CMA on lipid metabolism in the aged liver. Methods: 12-week-old and 88-week-old mice were employed to assess the effect of aging on hepatic CMA activity. We generated CMA-deficient mouse primary hepatocytes using siRNA for Lamp2a and liver-specific LAMP2A knockdown mice via adeno-associated viruses expressing short hairpin RNAs to investigate the influence of CMA on lipid metabolism. Results: We noted aging-induced progression toward fatty liver and a decrease in LAMP2A levels in total protein and lysosomes. The expression of genes associated with fatty acid oxidation was markedly downregulated in the aged liver, as verified in CMA-deficient mouse primary hepatocytes. In addition, the aged liver accumulated nuclear receptor corepressor 1 (NCoR1), a negative regulator of peroxisome proliferator-activated receptor α (PPARα). We found that Hsc70 binds to NCoR1 via the KFERQ motif. Lamp2a siRNA treatment accumulated NCoR1 and decreased the fatty acid oxidation rate. Pharmacological activation of CMA by AR7 treatment increased LAMP2A expression, leading to NCoR1 degradation. A liver-specific LAMP2A knockdown via adeno-associated viruses expressing short hairpin RNAs caused NCoR1 accumulation, inactivated PPARα, downregulated the expression of fatty acid oxidation-related genes and significantly increased liver triglyceride levels. Conclusions: Our results elucidated a novel PPARα regulatory mechanism involving CMA-mediated NCoR1 degradation during aging. These findings demonstrate that CMA dysregulation is crucial for the progression of aging-related fatty liver diseases.

Bcl-2 family kin (Bfk), also known as Bcl-2-like 15, plays an essential role in regulating apoptosis by eliciting weak pro-apoptotic responses in the gastrointestinal tract. Human Bfk is a novel Bcl-2 family protein owing to its unique domain composition involving BH2 and BH3. However, the molecular mechanism underlying the regulation of apoptosis by Bfk remains unclear. Here, we first report the crystal structure of human full-length Bfk. Surprisingly, the structure of Bfk adopts a canonical Bcl-2 fold but lacks the hydrophobic cleft, which could accommodate a BH3 domain from other Bcl-2 family proteins. Our biophysical interaction analysis proved that the full-length Bfk itself does not interact with multi-domain Bcl-2 family proteins or a BH3-containing peptide. Instead, Bfk is structurally and functionally reminiscent of Bid, a BH3-only protein in the Bcl-2 family, with similar conformations of helices α3-α5 and the specific motif in helix α5. Not only structural analyses of the full-length Bfk but also molecular dynamics simulation suggested that Bfk elicits its pro-apoptotic activity through a Bid-like apoptotic mechanism in which the BH3 domain is released upon caspase-mediated cleavage and a conformational change of the truncated form. Indeed, the BH3 peptide derived from Bfk exhibited in vitro interactions with Bcl-2, Bcl-XL, and Bak. These findings provide new insights into the molecular characteristics of Bfk and a valuable foundation for development of a new therapeutic target to control apoptosis.

Aminoacyl-tRNA synthetases (ARSs) play essential roles in protein biosynthesis as well as in other cellular processes, often using evolutionarily acquired domains. For possible cooperativity and synergistic effects, nine ARSs assemble into the multi-tRNA synthetase complex (MSC) with three scaffold proteins: aminoacyl-tRNA synthetase complex-interacting multifunctional proteins 1, 2 and 3 (AIMP1, AIMP2 and AIMP3). X-ray crystallographic methods were implemented in order to determine the structure of a ternary subcomplex of the MSC comprising aspartyl-tRNA synthetase (DRS) and two glutathione S-transferase (GST) domains from AIMP2 and glutamyl-prolyl-tRNA synthetase (AIMP2GST and EPRSGST, respectively). While AIMP2GST and EPRSGST interact via conventional GST heterodimerization, DRS strongly interacts with AIMP2GST via hydrogen bonds between the α7–β9 loop of DRS and the β2–α2 loop of AIMP2GST, where Ser156 of AIMP2GST is essential for the assembly. Structural analyses of DRS–AIMP2GST–EPRSGST reveal its pivotal architecture in the MSC and provide valuable insights into the overall assembly and conditionally required disassembly of the MSC.

Stabilized, native-like trimers of the HIV envelope protein, such as SOSIP trimers, are potential antigens for an HIV vaccine. Here, the authors generate a SOSIP trimer based on the consensus sequence of group M isolates, determine its structure and exposure of common epitopes, and show immunogenicity in rabbits and non-human primates.

Genome-wide association studies of gastric cancer (GC) cases have revealed common gastric cancer susceptibility loci with low effect size. We investigated rare variants with high effect size via whole-exome sequencing (WES) of subjects with familial clustering of gastric cancer. WES of DNAs from the blood of 19 gastric cancer patients and 36 unaffected family members from 14 families with two or more gastric cancer patients were tested. Linkage analysis combined with association tests were performed using Pedigree Variant Annotation, Analysis, and Search Tool (pVAAST) software. Based on the logarithm of odds (LOD) and permutation-based composite likelihood ratio test (CLRT) from pVAAST, MUC4 was identified as a predisposing gene (LOD P-value = 1.9×10-5; permutation-based P-value of CLRT ≤ 9.9×10-9). In a larger cohort consisting of 597 GC patients and 9,759 healthy controls genotyped with SNP array, we discovered common variants in MUC4 regions (rs148735556, rs11717039, and rs547775645) significantly associated with GC supporting the association of MUC4 with gastric cancer. And the MUC4 variants were found in higher frequency in The Cancer Genome Atlas Study (TCGA) germline samples of patients with multiple cancer types. Immunohistochemistry indicated that MUC4 was downregulated in the noncancerous gastric mucosa of subjects with MUC4 germline missense variants, suggesting that loss of the protective function of MUC4 predisposes an individual to gastric cancer. Rare variants in MUC4 can be novel gastric cancer susceptibility loci in Koreans possessing the familial clustering of gastric cancer.

A series of novel 5′(7′)-substituted-2′-oxospiro[1,3]dioxolane-2,3′-indoline-based N-hydroxypropenamides were designed, synthesized and evaluated for histone deacetylase (HDAC) inhibition and cytotoxicity. It was found that the compounds in this series displayed potent inhibitory effects against HDAC2 with IC50 values as low as 0.284 μM, almost comparable to that of SAHA (IC50, 0.265 μM), a positive control. In Western blot analysis, these compounds also exhibited noted inhibition toward histone deacetylation and this inhibition was found to correlate well with the cytotoxicity of the compounds in three human cancer cell lines. Docking studies indicated the compounds in this series bound to HDAC2 with high binding affinities (∼−9.8 kcal/mol) compared to SAHA (−7.4 kcal/mol).

Adiponectin is an adipocyte-derived cytokine having an insulin-sensitizing activity. During the phenotypic screening of secondary metabolites derived from the marine fungus Aspergillus terreus, a poly cyclin-dependent kinase (CDK) inhibitor butyrolactone I affecting CDK1 and CDK5 was discovered as a potent adiponectin production-enhancing compound in the adipogenesis model of human bone marrow-derived mesenchymal stem cells (hBM-MSCs). CDK5 inhibitors exhibit insulin-sensitizing activities by suppressing the phosphorylation of peroxisome proliferator-activated receptor γ (PPARγ). However, the adiponectin production-enhancing activities of butyrolactone I have not been correlated with the potency of CDK5 inhibitor activities. In a target identification study, butyrolactone I was found to directly bind to PPARγ. In the crystal structure of the human PPARγ, the ligand-binding domain (LBD) in complex with butyrolactone I interacted with the amino acid residues located in the hydrophobic binding pockets of the PPARγ LBD, which is a typical binding mode of the PPARγ partial agonists. Therefore, the adiponectin production-enhancing effect of butyrolactone I was mediated by its polypharmacological dual modulator activities as both a CDK5 inhibitor and a PPARγ partial agonist.

Arrest defective 1 (ARD1), also known as N(alpha)-acetyltransferase 10 (NAA10) was originally identified as an N-terminal acetyltransferase (NAT) that catalyzes the acetylation of N-termini of newly synthesized peptides. After that, mammalian ARD1/NAA10 expanded its’ role to lysine acetyltransferase (KAT) that post-translationally acetylates internal lysine residues of proteins. ARD1/NAA10 is the only enzyme with both NAT and KAT activities. However, recent studies on the role of human ARD1/NAA10 (hARD1/NAA10) in lysine acetylation are contradictory, as crystal structure and in vitro acetylation assay results revealed the lack of KAT activity. Thus, the role of hARD1/NAA10 in lysine acetylation is still debating. Here, we found a clue that possibly explains these complicated and controversial results on KAT activity of hARD1/NAA10. Recombinant hARD1/NAA10 exhibited KAT activity, which disappeared soon in vitro. Size-exclusion analysis revealed that most recombinant hARD1/NAA10 formed oligomers over time, resulting in the loss of KAT activity. While oligomeric recombinant hARD1/NAA10 lost its ability for lysine acetylation, its monomeric form clearly exhibited lysine acetylation activity in vitro. We also characterized the KAT activity of hARD1/NAA10 that was influenced by several experimental conditions, including concentration of reactants and reaction time. Taken together, our study proves that recombinant hARD1/NAA10 exhibits KAT activity in vitro but only under accurate conditions, including reactant concentrations and reaction duration.

The N-degron pathway is a proteolytic system in which a single N-terminal amino acid acts as a determinant of protein degradation. Especially, degradation signaling of N-terminal asparagine (Nt-Asn) in eukaryotes is initiated from its deamidation by N-terminal asparagine amidohydrolase 1 (NTAN1) into aspartate. Here, we have elucidated structural principles of deamidation by human NTAN1. NTAN1 adopts the characteristic scaffold of CNF1/YfiH-like cysteine hydrolases that features an α-β-β sandwich structure and a catalytic triad comprising Cys, His, and Ser. In vitro deamidation assays using model peptide substrates with varying lengths and sequences showed that NTAN1 prefers hydrophobic residues at the second-position. The structures of NTAN1-peptide complexes further revealed that the recognition of Nt-Asn is sufficiently organized to produce high specificity, and the side chain of the second-position residue is accommodated in a hydrophobic pocket adjacent to the active site of NTAN1. Collectively, our structural and biochemical analyses of the substrate specificity of NTAN1 contribute to understanding the structural basis of all three amidases in the eukaryotic N-degron pathway.

The N-Myc downstream-regulated gene (NDRG) family belongs to the α/β-hydrolase fold and is known to exert various physiologic functions in cell proliferation, differentiation, and hypoxia-induced cancer metabolism. In particular, NDRG3 is closely related to proliferation and migration of prostate cancer cells, and recent studies reported its implication in lactate-triggered hypoxia responses or tumorigenesis. However, the underlying mechanism for the functions of NDRG3 remains unclear. Here, we report the crystal structure of human NDRG3 at 2.2 Å resolution, with six molecules in an asymmetric unit. While NDRG3 adopts the α/β-hydrolase fold, complete substitution of the canonical catalytic triad residues to non-reactive residues and steric hindrance around the pseudo-active site seem to disable the α/β-hydrolase activity. While NDRG3 shares a high similarity to NDRG2 in terms of amino acid sequence and structure, NDRG3 exhibited remarkable structural differences in a flexible loop corresponding to helix α6 of NDRG2 that is responsible for tumor suppression. Thus, this flexible loop region seems to play a distinct role in oncogenic progression induced by NDRG3. Collectively, our studies could provide structural and biophysical insights into the molecular characteristics of NDRG3.

Cardiac glycosides (CGs) are natural compounds used traditionally to treat congestive heart diseases. Recent investigations repositioned CGs as potential anticancer agents. To discover novel cytotoxic CG scaffolds, we selected the cardenolide glucoevatromonoside (GEV) out of 46 CGs for its low nanomolar anti-lung cancer activity. GEV presented reduced toxicity toward non-cancerous cell types (lung MRC-5 and PBMC) and high-affinity binding to the Na+/K+-ATPase α subunit, assessed by computational docking. GEV-induced cell death was caspase-independent, as investigated by a multiparametric approach, and culminates in severe morphological alterations in A549 cells, monitored by transmission electron microscopy, live cell imaging and flow cytometry. This non-canonical cell death was not preceded or accompanied by exacerbation of autophagy. In the presence of GEV, markers of autophagic flux (e.g. LC3I-II conversion) were impacted, even in presence of bafilomycin A1. Cell death induction remained unaffected by calpain, cathepsin, parthanatos, or necroptosis inhibitors. Interestingly, GEV triggered caspase-dependent apoptosis in U937 acute myeloid leukemia cells, witnessing cancer-type specific cell death induction. Differential cell cycle modulation by this CG led to a G2/M arrest, cyclin B1 and p53 downregulation in A549, but not in U937 cells. We further extended the anti-cancer potential of GEV to 3D cell culture using clonogenic and spheroid formation assays and validated our findings in vivo by zebrafish xenografts. Altogether, GEV shows an interesting anticancer profile with the ability to exert cytotoxic effects via induction of different cell death modalities.

Glutathione (GSH) degradation plays an essential role in GSH homeostasis, which regulates cell survival, especially in cancer cells. Among human GSH degradation enzymes, the ChaC2 enzyme acts on GSH to form 5-l-oxoproline and Cys-Gly specifically in the cytosol. Here, we report the crystal structures of ChaC2 in two different conformations and compare the structural features with other known γ-glutamylcyclotransferase enzymes. The unique flexible loop of ChaC2 seems to function as a gate to achieve specificity for GSH binding and regulate the constant GSH degradation rate. Structural and biochemical analyses of ChaC2 revealed that Glu74 and Glu83 play crucial roles in directing the conformation of the enzyme and in modulating the enzyme activity. Based on a docking study of GSH to ChaC2 and binding assays, we propose a substrate-binding mode and catalytic mechanism. We also found that overexpression of ChaC2, but not mutants that inhibit activity of ChaC2, significantly promoted breast cancer cell proliferation, suggesting that the GSH degradation by ChaC2 affects the growth of breast cancer cells. Our structural and functional analyses of ChaC2 will contribute to the development of inhibitors for the ChaC family, which could effectively regulate the progression of GSH degradation-related cancers.

Imatinib is an effective anticancer drug for the treatment of leukemia. Interestingly, when an FDA-approved drug library was tested for agents that block peroxisome proliferator-activated receptor γ (PPARγ) phosphorylation at Ser245 to evaluate possibilities of antidiabetic drug repositioning, imatinib was determined as a PPARγ antagonist ligand. However, it is not well understood how imatinib binds to PPARγ or would improve insulin sensitivity without classical agonism. Here, we report the crystal structure of the PPARγ R288A mutant in complex with imatinib. Imatinib bound to Arm2 and Arm3 regions in the ligand-binding domain (LBD) of PPARγ, of which the Arm3 region is closely related to the inhibition of PPARγ phosphorylation at Ser245. The binding of imatinib in LBD induced a stable conformation of helix H2′ and the Ω loop compared with the ligand-free state. In contrast, imatinib does not interact with Tyr473 on PPARγ helix H12, which is important for the classical agonism associated with side effects. Our study provides new structural insights into the PPARγ regulation by imatinib and may contribute to the development of new antidiabetic drugs targeting PPARγ while minimizing known side effects.

Human endothelial-overexpressed lipopolysaccharide-associated factor 1 (EOLA1) has been suggested to regulate inflammatory responses in endothelial cells by controlling expression of proteins, interleukin-6 and vascular cell adhesion molecule-1, and by preventing apoptosis. To elucidate the structural basis of the EOLA1 function, we determined its crystal structure at 1.71 Å resolution and found that EOLA1 is structurally similar to an activating signal cointegrator-1 homology (ASCH) domain with a characteristic β-barrel fold surrounded by α-helices. Despite its low sequence identity with other ASCH domains, EOLA1 retains a conserved ‘GxKxxExR’ motif in its cavity structure. The cavity harbors aromatic and polar residues, which are speculated to accommodate nucleotide molecules as do YT521-B homology (YTH) proteins. Additionally, EOLA1 exhibits a positively charged cleft, similar to those observed in YTH proteins and the ASCH protein from Zymomonas mobilis that exerts ribonuclease activity. This implies that the positively charged cleft in EOLA1 could stabilize the binding of RNA molecules. Taken together, we suggest that EOLA1 controls protein expression through RNA binding to play protective roles against endothelial cell injuries resulting from lipopolysaccharide (LPS)-induced inflammation responses.

FolC plays important roles in the folate metabolism of cells by attaching l-Glu to dihydropteroate (DHP) and folate, which are known activities of dihydrofolate synthetase (DHFS) and folylpolyglutamate synthetase (FPGS), respectively. Here, we determined the crystal structure of Helicobacter pylori FolC (HpFolC) at 1.95 Å resolution using the single-wavelength anomalous diffraction method. HpFolC has globular N- and C-terminal domains connected by a single loop, and a binding site for ATP is located between the two domains. Apo-HpFolC was crystallized in the presence of citrate in a crystallization solution, which was held in the ATP-binding site. Structural motifs such as the P-loop and Ω-loop of HpFolC for binding of ATP and two magnesium ions are well conserved in spite of the low overall sequence similarity to other FolC/FPGSs. The Ω-loop would also recognize a folate molecule, and the DHP-binding loop of HpFolC is expected to exhibit a unique recognition mode on DHP, compared with other FolCs. Because human FolC is known to only exhibit FPGS activity, the DHFS activity of bacterial FolC is an attractive target for the eradication of pathogenic bacteria. Consequently, our structural analyses of HpFolC provide a valuable foundation for a universal antibacterial strategy against H. pylori as well as other pathogenic bacteria.

Treatment of acute myeloid leukemia (AML) patients is still hindered by resistance and relapse, resulting in an overall poor survival rate. Recently, combining specific B-cell lymphoma (Bcl)-2 inhibitors with compounds downregulating myeloid cell leukemia (Mcl)-1 has been proposed as a new effective strategy to eradicate resistant AML cells. We show here that 1(R), 6(S), 1’(R), 6’(S), 11(R), 17(S)-fistularin-3, a bromotyrosine compound of the fistularin family, isolated from the marine sponge Suberea clavata, synergizes with Bcl-2 inhibitor ABT-199 to efficiently kill Mcl-1/Bcl-2-positive AML cell lines, associated with Mcl-1 downregulation and endoplasmic reticulum stress induction. The absolute configuration of carbons 11 and 17 of the fistularin-3 stereoisomer was fully resolved in this study for the first time, showing that the fistularin we isolated from the marine sponge Subarea clavata is in fact the (+)-11(R), 17(S)-fistularin-3 stereoisomer keeping the known configuration 1(R), 6(S), 1’(R), and 6’(S) for the verongidoic acid part. Docking studies and in vitro assays confirm the potential of this family of molecules to inhibit DNA methyltransferase 1 activity.

NAD+-dependent histone deacetylases (sirtuins) are implicated in cellular processes such as proliferation, DNA repair, and apoptosis by regulating gene expression and the functions of numerous proteins. Due to their key role in cells, the discovery of small molecule sirtuin modulators has been of significant interest for diverse therapeutic applications. In particular, it has been shown that inhibition of sirtuin 1 and 2 activities is beneficial for cancer treatment. Here, we demonstrate that the fungal metabolite eurochevalierine from the fungus Neosartorya pseudofischeri inhibits sirtuin 1 and 2 activities (IC50 about 10 µM) without affecting sirtuin 3 activity. The binding modes of the eurochevalierine for sirtuin 1 and 2 have been identified through computational docking analyses. Accordingly, this sequiterpene alkaloid induces histone H4 and α-tubulin acetylation in various cancer cell models in which it induces strong cytostatic effects without affecting significantly the viability of healthy PBMCs. Importantly, eurochevalierine targets preferentially cancer cell proliferation (selectivity factor ≫ 7), as normal human primary CD34+ stem/progenitor cells were less affected by the treatment. Finally, eurochevalierine displays suitable drug-likeness parameters and therefore represent a promising scaffold for lead molecule optimization to study the mechanism and biological roles of sirtuins and potentially a basis for development into therapeutics.

Despite the growing attention given to Traditional Medicine (TM) worldwide, there is no well-known, publicly available, integrated bio-pharmacological Traditional Korean Medicine (TKM) database for researchers in drug discovery. In this study, we have constructed PharmDB-K, which offers comprehensive information relating to TKM-associated drugs (compound), disease indication, and protein relationships. To explore the underlying molecular interaction of TKM, we integrated fourteen different databases, six Pharmacopoeias, and literature, and established a massive bio-pharmacological network for TKM and experimentally validated some cases predicted from the PharmDB-K analyses. Currently, PharmDB-K contains information about 262 TKMs, 7,815 drugs, 3,721 diseases, 32,373 proteins, and 1,887 side effects. One of the unique sets of information in PharmDB-K includes 400 indicator compounds used for standardization of herbal medicine. Furthermore, we are operating PharmDB-K via phExplorer (a network visualization software) and BioMart (a data federation framework) for convenient search and analysis of the TKM network. Database URL: http://pharmdb-k.org, http://biomart.i-pharm.org.

Neural stem cells (NSCs) have the ability to proliferate and differentiate into neurons and glia. Regulation of NSC fate by small molecules is important for the generation of a certain type of cell. The identification of small molecules that can induce new neurons from NSCs could facilitate regenerative medicine and drug development for neurodegenerative diseases. In this study, we screened natural compounds to identify molecules that are effective on NSC cell fate determination. We found that Kuwanon V (KWV), which was isolated from the mulberry tree (Morus bombycis) root, increased neurogenesis in rat NSCs. In addition, during NSC differentiation, KWV increased cell survival and inhibited cell proliferation as shown by 5-bromo-2-deoxyuridine pulse experiments, Ki67 immunostaining and neurosphere forming assays. Interestingly, KWV enhanced neuronal differentiation and decreased NSC proliferation even in the presence of mitogens such as epidermal growth factor and fibroblast growth factor 2. KWV treatment of NSCs reduced the phosphorylation of extracellular signal-regulated kinase 1/2, increased mRNA expression levels of the cyclin-dependent kinase inhibitor p21, down-regulated Notch/Hairy expression levels and up-regulated microRNA miR-9, miR-29a and miR-181a. Taken together, our data suggest that KWV modulates NSC fate to induce neurogenesis, and it may be considered as a new drug candidate that can regenerate or protect neurons in neurodegenerative diseases.

Thermoplasma acidophilum is a thermophilic archaeon that uses both non-phosphorylative Entner-Doudoroff (ED) pathway and Embden-Meyerhof-Parnas (EMP) pathway for glucose degradation. While triosephosphate isomerase (TPI), a well-known glycolytic enzyme, is not involved in the ED pathway in T. acidophilum, it has been considered to play an important role in the EMP pathway. Here, we report crystal structures of apo- and glycerol-3-phosphate-bound TPI from T. acidophilum (TaTPI). TaTPI adopts the canonical TIM-barrel fold with eight α-helices and parallel eight β-strands. Although TaTPI shares ~30% sequence identity to other TPIs from thermophilic species that adopt tetrameric conformation for enzymatic activity in their harsh physiological environments, TaTPI exists as a dimer in solution. We confirmed the dimeric conformation of TaTPI by analytical ultracentrifugation and size-exclusion chromatography. Helix 5 as well as helix 4 of thermostable tetrameric TPIs have been known to play crucial roles in oligomerization, forming a hydrophobic interface. However, TaTPI contains unique charged-amino acid residues in the helix 5 and adopts dimer conformation. TaTPI exhibits the apparent Td value of 74.6°C and maintains its overall structure with some changes in the secondary structure contents at extremely acidic conditions (pH 1-2). Based on our structural and biophysical analyses of TaTPI, more compact structure of the protomer with reduced length of loops and certain patches on the surface could account for the robust nature of Thermoplasma acidophilum TPI.

The N-end rule states that half-life of protein is determined by their N-terminal amino acid residue. N-terminal glutamine amidohydrolase (Ntaq) converts N-terminal glutamine to glutamate by eliminating the amine group and plays an essential role in the N-end rule pathway for protein degradation. Here, we report the crystal structure of human Ntaq1 bound with the N-terminus of a symmetry-related Ntaq1 molecule at 1.5 Å resolution. The structure reveals a monomeric globular protein with alpha-beta-alpha three-layer sandwich architecture. The catalytic triad located in the active site, Cys-His-Asp, is highly conserved among Ntaq family and transglutaminases from diverse organisms. The N-terminus of a symmetry-related Ntaq1 molecule bound in the substrate binding cleft and the active site suggest possible substrate binding mode of hNtaq1. Based on our crystal structure of hNtaq1 and docking study with all the tripeptides with N-terminal glutamine, we propose how the peptide backbone recognition patch of hNtaq1 forms nonspecific interactions with N-terminal peptides of substrate proteins. Upon binding of a substrate with N-terminal glutamine, active site catalytic triad mediates the deamination of the N-terminal residue to glutamate by a mechanism analogous to that of cysteine proteases.

The influenza virus surface glycoprotein hemagglutinin (HA) is responsible for viral attachment to sialic acid-containing host cell receptors and it facilitates the initial stage of viral infection. In the present study, we isolated an RNA aptamer specific to the glycosylated receptor-binding domain of the HA protein (gHA1) after 12 cycles of the systematic evolution of ligands by exponential enrichment procedure (SELEX), and we then investigated if the selected aptamer suppresses viral infection in host cells. Nitrocellulose filter binding and enzyme-linked immunosorbent assay (ELISA) experiments revealed that 1 RNA aptamer, HA12-16, bound specifically to the gHA1 protein. Cell viability assay showed that the HA12-16 RNA aptamer suppressed viral infection in host cells by enhancing cell viability. Immunofluorescence microscopic analysis further demonstrated that the HA12-16 RNA aptamer suppresses viral attachment to host cells by neutralizing the receptor-binding site of influenza virus HA. These results indicate that the isolated RNA aptamer can be developed as an antiviral reagent against influenza through appropriate therapeutic formulation.