1.Rational design of the C-terminal Loop region of leucine dehydrogenase and cascade biosynthesis L-2-aminobutyric acid.
Jiajie CHEN ; Meijuan XU ; Taowei YANG ; Xian ZHANG ; Minglong SHAO ; Huazhong LI ; Zhiming RAO
Chinese Journal of Biotechnology 2021;37(12):4254-4265
Leucine dehydrogenase (LDH) is the key rate-limiting enzyme in the production of L-2-aminobutyric acid (L-2-ABA). In this study, we modified the C-terminal Loop region of this enzyme to improve the specific enzyme activity and stability for efficient synthesis of L-2-ABA. Using molecular dynamics simulation of LDH, we analyzed the change of root mean square fluctuation (RMSF), rationally designed the Loop region with greatly fluctuated RMSF, and obtained a mutant EsLDHD2 with a specific enzyme activity 23.2% higher than that of the wild type. Since the rate of the threonine deaminase-catalyzed reaction converting L-threonine into 2-ketobutyrate was so fast, the multi-enzyme cascade catalysis system became unbalanced. Therefore, the LDH and the formate dehydrogenase were double copied in a new construct E. coli BL21/pACYCDuet-RM. Compared with E. coli BL21/pACYCDuet-RO, the molar conversion rate of L-2-ABA increased by 74.6%. The whole cell biotransformation conditions were optimized and the optimal pH, temperature and substrate concentration were 7.5, 35 °C and 80 g/L, respectively. Under these conditions, the molar conversion rate was higher than 99%. Finally, 80 g and 40 g L-threonine were consecutively fed into a 1 L reaction mixture under the optimal conversion conditions, producing 97.9 g L-2-ABA. Thus, this strategy provides a green and efficient synthesis of L-2-ABA, and has great industrial application potential.
Aminobutyrates
;
Escherichia coli/genetics*
;
Leucine Dehydrogenase/genetics*
;
Threonine Dehydratase
2.Leucine-rich repeats containing 4 protein (LRRC4) in memory, psychoneurosis, and glioblastoma.
Chinese Medical Journal 2023;136(1):4-12
Leucine-rich repeats containing 4 ( LRRC4 , also named netrin-G ligand 2 [NGL-2]) is a member of the NetrinGs ligands (NGLs) family. As a gene with relatively high and specific expression in brain, it is a member of the leucine-rich repeat superfamily and has been proven to be a suppressor gene for gliomas, thus being involved in gliomagenesis. LRRC4 is the core of microRNA-dependent multi-phase regulatory loops that inhibit the proliferation and invasion of glioblastoma (GB) cells, including LRRC4/NGL2-activator protein 2 (AP2)-microRNA (miR) 182-LRRC4 and LRRC4-miR185-DNA methyltransferase 1 (DNMT1)-LRRC4/specific protein 1 (SP1)-DNMT1-LRRC4. In this review, we demonstrated LRRC4 as a new member of the partitioning-defective protein (PAR) polarity complex that promotes axon differentiation, mediates the formation and plasticity of synapses, and assists information input to the hippocampus and storage of memory. As an important synapse regulator, aberrant expression of LRRC4 has been detected in autism, spinal injury and GBs. LRRC4 is a candidate susceptibility gene for autism and a neuro-protective factor in spinal nerve damage. In GBs, LRRC4 is a novel inhibitor of autophagy, and an inhibitor of protein-protein interactions involving in temozolomide resistance, tumor immune microenvironment, and formation of circular RNA.
Humans
;
Cell Line, Tumor
;
Glioblastoma/metabolism*
;
Leucine
;
Leucine-Rich Repeat Proteins/genetics*
;
MicroRNAs
;
Nerve Tissue Proteins/genetics*
;
Tumor Microenvironment
3.Serological Characteristics and Molecular Biological Mechanism of AEL.02 Subtype.
Feng-Wu QIU ; Xiao-Ling SHI ; Mei-Hua LI ; Gang SHEN
Journal of Experimental Hematology 2022;30(5):1562-1566
OBJECTIVE:
To explore the serological characteristics and molecular biological mechanism of an ael subtype specimen.
METHODS:
The ABO blood typing was identified by routine blood group serological and absorption/elution methods; PCR-SBT method for ABO genotyping: 7 exons of ABO gene were amplified by PCR, the amplified products were purified, and then sequencing primers were designed and the amplified products were sequenced directly for analysis; 3D molecular model was constructed and the difference of free energy (ΔΔG) was used to predict the GTA mutant stability.
RESULTS:
A antigen was not detected on erythrocytes through absorption and elution tests, which was not consistent with the serological characteristics of ael, and the serological typing results were ambiguous. The ABO genotype was ABO*AEL.02/O.01.01, and there were two mutations in exon 7 of the gene, c.467C>T and c.646T>A, which could lead to the replacement of proline with leucine at position 156 (p.Pro156Leu) and phenylalanine with isoleucine at position 216 on the GTA, respectively. The 3D model predicts that the mutations do not introduce new hydrogen bonds to the GTA mutant and do not form a new secondary structure, but can lead to an increase in the ΔΔG value of the GTA mutant, suggesting a decrease in protein stability.
CONCLUSION
The serological characteristics alone is not reliable to determine the ael subype; the ael phenotype may be due to the GTA mutant that reduces enzyme stability.
ABO Blood-Group System/genetics*
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Alleles
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Genotype
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Isoleucine/genetics*
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Leucine/genetics*
;
Phenotype
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Phenylalanine/genetics*
;
Proline/genetics*
4.Altered Motor Performance, Sleep EEG, and Parkinson's Disease Pathology Induced by Chronic Sleep Deprivation in Lrrk2G2019S Mice.
Xinyao LIU ; Hang YU ; Yuanyuan WANG ; Song LI ; Cheng CHENG ; Murad AL-NUSAIF ; Weidong LE
Neuroscience Bulletin 2022;38(10):1170-1182
Parkinson's disease (PD) is a multifaceted disease in which environmental variables combined with genetic predisposition cause dopaminergic (DAergic) neuron loss in the substantia nigra pars compacta. The mutation of leucine-rich repeat kinase 2 (Lrrk2) is the most common autosomal dominant mutation in PD, and it has also been reported in sporadic cases. A growing body of research suggests that circadian rhythm disruption, particularly sleep-wake abnormality, is common during the early phase of PD. Our present study aimed to evaluate the impact of sleep deprivation (SD) on motor ability, sleep performance, and PD pathologies in Lrrk2G2019S transgenic mice. After two months of SD, Lrrk2G2019S mice at 12 months of age showed an exacerbated PD-like phenotype with motor deficits, a reduced striatal DA level, degenerated DAergic neurons, and altered sleep structure and biological rhythm accompanied by the decreased protein expression level of circadian locomotor output cycles kaput Lrrk2 gene in the brain. All these changes persisted and were even more evident in 18-month-old mice after 6 months of follow-up. Moreover, a significant increase in α-synuclein aggregation was found in SD-treated transgenic mice at 18 months of age. Taken together, our findings indicate that sleep abnormalities, as a risk factor, may contribute to the pathogenesis and progression of PD. Early detection of sleep disorders and improvement of sleep quality may help to delay disease progression and provide long-term clinical benefits.
Animals
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Electroencephalography
;
Leucine/genetics*
;
Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/genetics*
;
Mice
;
Mice, Transgenic
;
Mutation
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Parkinson Disease/metabolism*
;
Sleep Deprivation/complications*
;
alpha-Synuclein/genetics*
5.The role and mechanism of leucyl-tRNA synthetase in the regulation of protein synthesis in aging skeletal muscle.
Zhi XIA ; Hua-Yu SHANG ; Qian-Jin WANG ; Yan ZHAO ; Xiao-Min DING
Acta Physiologica Sinica 2020;72(4):523-531
The imbalance of protein metabolism is the major cause of skeletal muscle atrophy, and the decrease of protein synthesis directly leads to the occurrence and development of age-related sarcopenia. The canonical role of leucyl-tRNA synthetase (LeuRS) is ligating leucine to the cognate tRNA, and thus it plays a central role in genetic coding. With the further studies of LeuRS in recent years, LeuRS has been found to control protein homeostasis in aging skeletal muscle via its non-canonical role. In this paper, we reviewed the structure and biological features of aminoacyl-tRNA synthetase and LeuRS, and summarized the recent advances in studies on the effects of LeuRS in regulating aging skeletal muscle protein synthesis as an intracellular leucine sensor. Moreover, we also analyzed the potential role of LeuRS in activation of mammalian target of rapamycin complex 1 (mTORC1) signaling transduction pathway in response to anabolic stimuli such as exercise and amino acids ingestion. This paper may provide some new ideas for the prevention, diagnosis and treatment of age-related sarcopenia.
Amino Acyl-tRNA Synthetases
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genetics
;
Leucine-tRNA Ligase
;
genetics
;
Muscle, Skeletal
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Protein Biosynthesis
6.Progress of TSC-22 gene research.
Journal of Central South University(Medical Sciences) 2011;36(7):592-596
Transformation growth factor β -inducible gene 22 (TSC-22) is a putative negative growth regulation and tumor suppressor gene. It has the ability to combine with other transcription factors to regulate the cell growth and apoptosis. TSC-22 is lowly expressed in many types of tumors,which may be related to the tumorgenesis and development.
Animals
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Genes, Tumor Suppressor
;
Humans
;
Leucine Zippers
;
genetics
;
Neoplasms
;
physiopathology
;
Repressor Proteins
;
genetics
;
physiology
;
Transcription Factors
7.A family case of beta-thalassemia minor and hemoglobin Queens: alpha 34 (B15) Leu-Arg.
Nam Yong LEE ; Han Ik CHO ; Sang In KIM ; Byoung Kook KIM ; Yuzo OHBA ; Yukio HATTORI
Journal of Korean Medical Science 1992;7(4):385-388
We report a Korean family case of beta-thalassemia minor and Hb Queens. This is the first case report of Hb Queens in Korea. A 43-year-old male and his four family members had beta-thalassemia minor which is very rare in Korea. Incidentally, an alpha chain variant with a high isoelectric point was also found in two other family members without clinical problems and was finally identified as alpha 34 (B15) Leu-Arg or Hemoglobin Queens.
Adult
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Arginine/genetics
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Female
;
Hemoglobins, Abnormal/*genetics
;
Humans
;
Korea
;
Leucine/genetics
;
Male
;
Pedigree
;
beta-Thalassemia/blood/*genetics
8.Synthesis of L-2-aminobutyric acid by leucine dehydrogenase coupling with an NADH regeneration system.
Likun ZHANG ; Yanming XIAO ; Weihua YANG ; Chao HUA ; Yun WANG ; Jingya LI ; Taowei YANG
Chinese Journal of Biotechnology 2020;36(5):992-1001
In this study, Escherichia coli BL21 (DE3) was used as the host to construct 2 recombinant E. coli strains that co-expressed leucine dehydrogenase (LDH, Bacillus cereus)/formate dehydrogenase (FDH, Ancylobacter aquaticus), or leucine dehydrogenase (LDH, Bacillus cereus)/alcohol dehydrogenase (ADH, Rhodococcus), respectively. L-2-aminobutyric acid was then synthesized by L-threonine deaminase (L-TD) with LDH-FDH or LDH-ADH by coupling with two different NADH regeneration systems. LDH-FDH process and LDH-ADH process were optimized and compared with each other. The optimum reaction pH of LDH-FDH process was 7.5, and the optimum reaction temperature was 35 °C. After 28 h, the concentration of L-2-aminobutyric acid was 161.8 g/L with a yield of 97%, when adding L-threonine in batches for controlling 2-ketobutyric acid concentration less than 15 g/L and using 50 g/L ammonium formate, 0.3 g/L NAD+, 10% LDH-FDH crude enzyme solution (V/V) and 7 500 U/L L-TD. The optimum reaction pH of LDH-ADH process was 8.0, and the optimum reaction temperature was 35 °C. After 24 h, the concentration of L-2-aminobutyric acid was 119.6 g/L with a yield of 98%, when adding L-threonine and isopropanol (1.2 times of L-threonine) in batches for controlling 2-ketobutyric acid concentration less than 15 g/L, removing acetone in time and using 0.3 g/L NAD⁺, 10% LDH-ADH crude enzyme solution (V/V) and 7 500 U/L L-TD. The process and results used in this paper provide a reference for the industrialization of L-2-aminobutyric acid.
Aminobutyrates
;
metabolism
;
Escherichia coli
;
genetics
;
Formate Dehydrogenases
;
metabolism
;
Leucine Dehydrogenase
;
metabolism
;
NAD
;
metabolism
9.Role of post-translational modification of basic leucine zipper transcription factors in response to abiotic stresses in plants.
Ying LI ; Weidi ZHAO ; Jinghua YANG ; Jiaqi LI ; Songyang HAN ; Yuekun REN ; Changhong GUO
Chinese Journal of Biotechnology 2024;40(1):53-62
Abiotic stresses substantially affect the growth and development of plants. Plants have evolved multiple strategies to cope with the environmental stresses, among which transcription factors play an important role in regulating the tolerance to abiotic stresses. Basic leucine zipper transcription factors (bZIP) are one of the largest gene families. The stability and activity of bZIP transcription factors could be regulated by different post-translational modifications (PTMs) in response to various intracellular or extracellular stresses. This paper introduces the structural feature and classification of bZIP transcription factors, followed by summarizing the PTMs of bZIP transcription factors, such as phosphorylation, ubiquitination and small ubiquitin-like modifier (SUMO) modification, in response to abiotic stresses. In addition, future perspectives were prospected, which may facilitate cultivating excellent stress-resistant crop varieties by regulating the PTMs of bZIP transcription factors.
Basic-Leucine Zipper Transcription Factors/genetics*
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Protein Processing, Post-Translational
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Phosphorylation
;
Transcription Factors/genetics*
;
Stress, Physiological/genetics*
10.Expression and Significance of Leucine-rich Repeat-containing G-protein Coupled Receptor 5/6 in Wnt Pathway in Children with Acute Lymphoblastic Leukemia.
Xuan LI ; Wen-Peng WANG ; Min ZHOU ; Xiao-Rui XU ; Ji-Zhao GAO
Acta Academiae Medicinae Sinicae 2021;43(4):595-602
Objective To study the expression and significance of leucine-rich repeat-containing G-protein coupled receptor(LGR)5/6 in childhood acute lymphoblastic leukemia(ALL). Methods A total of 39 children who had ALL and achieved complete remission on day 33 after induction therapy were enrolled.The children before induction therapy were considered as the incipient group,and those who achieved complete remission on day 33 by induction therapy were considered as the remission group.According to the degree of risk,they were assigned into 3 groups:low-risk(
Child
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Humans
;
Leucine
;
Precursor Cell Lymphoblastic Leukemia-Lymphoma
;
RNA, Messenger/genetics*
;
Receptors, G-Protein-Coupled/genetics*
;
Wnt Signaling Pathway