Korea Research Institute of Bioscience and Biotechnology
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Structural and biophysical analyses of human N-Myc downstream-regulated gene 3 (NDRG3) protein
No full textThe 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 A 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.
Machine learning classifies core and outer fucosylation of N-glycoproteins using mass spectrometry
No full textProtein glycosylation is known to be involved in biological progresses such as cell recognition, growth, differentiation, and apoptosis. Fucosylation of glycoproteins plays an important role for structural stability and function of N-linked glycoproteins. Although many of biological and clinical studies of protein fucosylation by fucosyltransferases has been reported, structural classification of fucosylated N-glycoproteins such as core or outer isoforms remains a challenge. Here, we report for the first time the classification of N-glycopeptides as core- and outer-fucosylated types using tandem mass spectrometry (MS/MS) and machine learning algorithms such as the deep neural network (DNN) and support vector machine (SVM). Training and test sets of more than 800 MS/MS spectra of N-glycopeptides from the immunoglobulin gamma and alpha 1-acid-glycoprotein standards were selected for classification of the fucosylation types using supervised learning models. The best-performing model had an accuracy of more than 99% against manual characterization and area under the curve values greater than 0.99, which were calculated by probability scores from target and decoy datasets. Finally, this model was applied to classify fucosylated N-glycoproteins from human plasma. A total of 82N-glycopeptides, with 54 core-, 24 outer-, and 4 dual-fucosylation types derived from 54 glycoproteins, were commonly classified as the same type in both the DNN and SVM. Specifically, outer fucosylation was dominant in tri- and tetra-antennary N-glycopeptides, while core fucosylation was dominant in the mono-, bi-antennary and hybrid types of N-glycoproteins in human plasma. Thus, the machine learning methods can be combined with MS/MS to distinguish between different isoforms of fucosylated N-glycopeptides.
A novel X-linked variant of IQSEC2 is associated with Lennox-gastaut syndrome and mild intellectual disability in three generations of a korean family
No full textAim: Lennox-Gastaut syndrome (LGS) is a severe type of childhood-onset epilepsy with multiple types of seizures, specific discharges on electroencephalography, and intellectual disability. However, LGS-related genes are largely unknown. To identify causative genes related to LGS, we collected and analyzed data from a three-generation Korean family in which one member had LGS and two had intellectual disability. Methods: Genomic DNAs were extracted from blood samples of all participants and used in whole-exome sequencing (WES). Genetic variants were detected by the Genome Analysis Toolkit and confirmed by Sanger sequencing. Variant pathogenicity was evaluated by prediction programs and the American College of Medical Genetics criteria. The LGS patient had generalized slow spike-and-wave discharges, multiple types of seizures, and developmental delay. Results: Analyses of the WES data from the family revealed a novel variant (c.1048G>A, p.Ala350Thr) in the IQ motif and Sec7 domain 2 (IQSEC2). This variant is within a highly evolutionarily conserved IQ-like motif, indicating a decrease in the calmodulin-binding capacity or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid transmission. The hemizygous variant in the male with LGS was a maternally inherited X-linked variant from the heterozygous maternal grandmother and mother, both of whom had intellectual disability. Conclusion: These findings indicate that the variant of IQSEC2 triggered both LGS and intellectual disability dependent on sex in this family. We report a novel X-linked inherited IQSEC2 variant for LGS and intellectual disability, which enhances the spectrum of variants in the IQ-like motif of IQSEC2.
Crystal structure of the Csm5 subunit of the type III-A CRISPR-Cas system
No full textThe Csm complex eliminates foreign RNA and DNA in the microbial defense CRISPR-Cas system. Csm5, one of the five subunits in the complex, facilitates crRNA maturation and target RNA binding in the type III system. However, the exact functional mechanism of Csm5 has remained elusive. Here, we report the crystal structure of the apo form of the Csm5 subunit at a resolution of 2.6 A. Structural comparison of amino acids in the complex bound to RNA exhibits notable conformational changes in the crRNA and the target RNA binding sites. Shifts in the β-hairpin motif (β5-β6), α13 helix (resides 352-383), and G-rich loop (residues 335-337) in the C-terminal domain indicate an induced movement by crRNA binding. The positively charged residues (Lys 92, Arg 95 and Lys 96) located in the β-α4 loop of the target RNA interface show high conformational flexibility, while three-helix bundles (α1-α3) of the N-domain involved in Csm2 binding exhibit a rotational shift. The altered architecture of the Csm5 subunit demonstrates remarkable versatility of the ferredoxin-like fold in the RNA binding protein and provides a structural basis for the mechanism for crRNA and target RNA binding in the type III-A Crispr-Cas system.
Engineering biology to construct microbial chassis for the production of difficult-to-express proteins
No full textA large proportion of the recombinant proteins manufactured today rely on microbe-based expression systems owing to their relatively simple and cost-effective production schemes. However, several issues in microbial protein expression, including formation of insoluble aggregates, low protein yield, and cell death are still highly recursive and tricky to optimize. These obstacles are usually rooted in the metabolic capacity of the expression host, limitation of cellular translational machineries, or genetic instability. To this end, several microbial strains having precisely designed genomes have been suggested as a way around the recurrent problems in recombinant protein expression. Already, a growing number of prokaryotic chassis strains have been genome-streamlined to attain superior cellular fitness, recombinant protein yield, and stability of the exogenous expression pathways. In this review, we outline challenges associated with heterologous protein expression, some examples of microbial chassis engineered for the production of recombinant proteins, and emerging tools to optimize the expression of heterologous proteins. In particular, we discuss the synthetic biology approaches to design and build and test genome-reduced microbial chassis that carry desirable characteristics for heterologous protein expression.
Sinirhodobacter hankyongi sp. nov., a novel denitrifying bacterium isolated from sludge
No full textA novel Gram-reaction-negative, facultative-aerobic, motile, non-spore-forming, oval-shaped and denitrifying bacterium, designated BO-81T, was isolated from sludge sampled in the Republic of Korea. This bacterium was investigated via a polyphasic approach to reveal its taxonomic position. The results of phylogenetic analysis based on 16S rRNA gene sequencing indicated that strain BO-81T belonged to the family Rhodobacteraceae and was related to the closest species Sinirhodobacter ferrireducens (98.8？% sequence similarity), 'Sinorhodobacter hungdaonensis' (98.4 %), Rhodobacter lacus (97.8？%), Sinorhodobacter populi (96.8？%) and Rhodobacter maris (96.2？%). The average nucleotide identity and DNA-DNA hybridization values between strain BO-81T and its closely related strains were 77.7-94.2？% and 20.1-55.9？%, respectively, indicating that BO-81T represents a novel species of the genus Sinirhodobacter. Growth occurred at 18-40？°C on Reasoner's 2A medium in the presence of 0-7？%？NaCl (w/v) and at pH 6.0-9.0. Strain BO-81T was characterized chemotaxonomically as having ubiquinone 10 as its predominant respiratory quinone, summed feature 8 (C18？:？1 ω6c and/or C18？:？1 ω7c) as its major fatty acid and phosphatidylglycerol and phosphatidylethanolamine as its predominant polar lipids. The G+C content of the genomic DNA was 68.3？mol%. The results of physiological and biochemical tests allowed phenotypic differentiation of strain BO-81T from other Sinirhodobacter species with validly published names. Therefore, the isolate represented a novel species, for which the name Sinirhodobacter hankyongi sp. nov. (type strain BO-81T=KACC 19677T=LMG 30808T) is proposed.
Brevundimonas fluminis sp. nov., isolated from a river
No full textA Gram-stain-negative, rod-shaped, motile, facultatively aerobic and ivory-pigmented bacterium (designated strain LA-55T) was isolated from a river in the Republic of Korea. On the basis of 16S rRNA gene sequencing, strain LA-55T clustered with species of the genus Brevundimonas and was closely related to B revundimonas kwangchunensis KSL-102T (97.3 %), B revundimonas aurantiaca DSM 4731T (97.1 %), B revundimonas albigilva NHI-13T (97.0 %), B revundimonas balnearis FDRGB2bT (97.0 %) and Brevundimonas aveniformis DSM 17977T (97.0 %). The average nucleotide identity value between strain LA-55T and its closest-related strain was 74.1 %, indicating that strain LA-55T represents a novel species of the genus Brevundimonas. Growth occurred at 15-40 °C on Reasoner's 2A medium in the presence of 0-2 % NaCl (w/v) and at pH 6.0-8.0. The genomic DNA G+C content was 70.5 mol% and ubiquinone 10 (Q-10) was the major respiratory quinone. The major cellular fatty acids (>5 %) were C1 8 :1 ω6c and/or C1 8 :1 ω7c (summed feature 8), C16 : 0, C1 6 :1 ω6c and/or C1 6 :1 ω7c (summed feature 3) and C18 : 1 ω7c 11-methyl. The polar lipids consisted of phosphatidylglycerol, 1,2-di-O-acyl-3-O-[d-glucopyranosyl-(1→4)-α-d-glucopyranuronosyl]glycerol, 1,2-di-O-acyl-3-O-α-d-glucopyranuronosyl glycerol, unidentified aminolipid, unidentified phosphoglycolipid and unidentified lipids. Physiological and biochemical characteristics indicated that strain LA-55T represents a novel species of the genus Brevundimonas, for which the name Brevundimonas fluminis sp. nov. is proposed. The type strain is LA-55T (=KACC 19639T=LMG 30850T).
Molecular cloning and functional characterization of a sweetpotato chloroplast IbDHAR3 gene in response to abiotic stress
No full textDehydroascorbate reductase (DHAR) plays a critical role in the regeneration of ascorbic acid (AsA), and widely involved in plant tolerance to biotic and abiotic stresses. In this study, the IbDHAR3 gene was cloned from sweetpotato cultivar Xushu 18 by RT-PCR. The full-length of this gene was 813 bp which encodes 270 amino acids. The IbDHAR3 protein contained two conserved domains of glutathione S-transferase (GST) and GST-C-DHAR, and one chloroplast transit peptide with 52 amino acids length. Transient expression in tobacco leaf epidermal cells indicated that IbDHAR3 protein is subcellular localized to chloroplast. The qRT-PCR results revealed that the relative expression level of IbDHAR3 in leaves is much higher than that in other tissues, and could be up-regulated by ABA, drought, salinity, and high-temperature stresses. The seed germination rate and root elongation were increased in contrast to wild type under mannitol and NaCl stresses in T3 transgenic Arabidopsis overexpressing IbDHAR3 gene. The soil drought experiments showed that the overexpression of IbDHAR3 gene in Arabidopsis reduced the malondialdehyde (MDA) and the H2O2 content, enhanced the level of AtGR gene expression, superoxide dismutase (SOD), ascorbate peroxidase (APX), DHAR activity, and the AsA content. Therefore, overexpression of IbDHAR3 gene could enhance the ability of scavenging reactive oxygen species such as H2O2 by promoting AsA-glutathione cycle and related antioxidant enzymes system, thereby contributing to increased stress tolerance in Arabidopsis.
IDH1-dependent α-KG regulates brown fat differentiation and function by modulating histone methylation
No full textOBJECTIVE:
Brown adipocytes play important roles in the regulation of energy homeostasis by uncoupling protein 1-mediated non-shivering thermogenesis. Recent studies suggest that brown adipocytes as novel therapeutic targets for combating obesity and associated diseases, such as type II diabetes. However, the molecular mechanisms underlying brown adipocyte differentiation and function are not fully understood.
METHODS:
We employed previous findings obtained through proteomic studies performed to assess proteins displaying altered levels during brown adipocyte differentiation. Here, we performed assays to determine the functional significance of their altered levels during brown adipogenesis and development.
RESULTS:
We identified isocitrate dehydrogenase 1 (IDH1) as upregulated during brown adipocyte differentiation, with subsequent investigations revealing that ectopic expression of IDH1 inhibited brown adipogenesis, whereas suppression of IDH1 levels promoted differentiation of brown adipocytes. Additionally, Idh1 overexpression resulted in increased levels of intracellular α-ketoglutarate (α-KG) and inhibited the expression of genes involved in brown adipogenesis. Exogenous treatment with α-KG reduced brown adipogenesis during the early phase of differentiation, and ChIP analysis revealed that IDH1-mediated α-KG reduced trimethylation of histone H3 lysine 4 in the promoters of genes associated with brown adipogenesis. Furthermore, administration of α-KG decreased adipogenic gene expression by modulating histone methylation in brown adipose tissues of mice.
CONCLUSION:
These results suggested that the IDH1-α-KG axis plays an important role in regulating brown adipocyte differentiation and might represent a therapeutic target for treating metabolic diseases.
Chronic infiltration of T lymphocytes into the brain in a non-human primate model of Parkinson’s disease
No full textStudy of interactions between the nervous system and immunity offers insights into the pathogenesis of Parkinson's disease (PD) and potential therapeutic strategies for neurodegenerative diseases. Studies on rodents have revealed regulatory mechanisms of microglial activation and T lymphocyte recruitment in PD. However, the mechanisms underlying chronic T lymphocyte infiltration into the brain after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) injection into a non-human primate (NHP) model of PD remain unknown. This study aimed to investigate changes in serum RANTES (regulated on activation, normal T cell expression and secretion) and analyze the chronic infiltration of T lymphocytes into the brain and microglia activation in NHPs at 48？weeks post-MPTP administration. We found selective and local chronic infiltration of CD4+ and CD8+ T lymphocytes, loss of dopaminergic neurons, dopamine transporter expression, chronic normalization of RANTES in the peripheral blood, and altered microglial morphology at 48？weeks after MPTP injection. This study confirms the involvement of CD4+ and CD8+ T lymphocyte infiltration in MPTP-induced NHP models of PD. Additionally, we corroborated previous findings regarding the mechanisms of T lymphocyte-induced neurodegeneration. The findings of chronic infiltration of T lymphocytes in our NHP model of PD provide novel insights into PD pathogenesis and the development of preventive and therapeutic agents.