1.Recombinant Expression and Purification of F-box and Leucine-rich Repeat Protein 5 (FBXL5) using a Prokaryotic Expression System
Fatima Joy C. Cruz ; Tomohide Saio ; Takeshi Uchida ; Koichiro Ishimori
Philippine Journal of Health Research and Development 2022;26(CAS Issue):41-57
Background:
The F-box and Leucine-rich Repeat Protein 5 (FBXL5), a member of the E3 ligases, is considered to be the central iron sensor in mammals. The cryo-EM structure of FBXL5 in complex with IRP2 and SKP1 was reported by Wang et.al. in 2020. Surprisingly, a 2Fe-2S cluster seemed to be responsible for the iron-sensing capability of FBXL5.
Objectives:
To further explore the mechanism of its regulation, it is important to study the interaction of FBXL5 with other proteins under regulated conditions so we attempted to express FBXL5 in the hopes of studying its interaction with IRPs in vitro.
Methodology:
Plasmids were constructed to express FBXL5 in Escherichia coli expression hosts. Purification of an MBP-fused FBXL5 and GST-fused FBXL5 were performed using affinity chromatography. Peptide Mass Fingerprinting, Circular Dichroism spectroscopy, and SEC-MALS were employed to analyze the purified MBP-FBXL5. GST-FBXL5 was also used in a pull-down assay with Iron Regulatory Protein 1 (IRP1).
Results:
We are successful in expressing and partially purifying full-length FBXL5 using E. coli with the aid of a protein tag, the maltose binding protein (MBP) tag. However, cleavage of the protein tag resulted in decreased stability of FBXL5 as shown in SEC-MALS data. CD spectroscopy showed consistent secondary structure of FBXL5. A preliminary pull-down assay of GST-FBXL5 with IRP1 showed that IRP1 displayed interaction with the recombinant GST-FBXL5.
Conclusion
FBXL5, a 78-kDa mammalian protein was overexpressed in a prokaryotic expression system made stable by a fusion protein. The interaction of GST-FBXL5 with IRP1 also shows that it is possible to study their interaction in vitro.
Leucine-Rich Repeat Proteins
2.Identification of intestine direct targets of Shouhui Tongbian Capsules using "target fishing" strategy.
Qiang GUO ; Lu YAO ; Zhong LIU ; Jing-Chun YAO ; Peng-Fei TU ; Ke-Wu ZENG
China Journal of Chinese Materia Medica 2021;46(3):505-510
"Target fishing" strategy was used to investigate the direct targets and mechanism of Shouhui Tongbian Capsules on relaxing bowel. Magnetic beads cross-linked with the chemical constituents from Shouhui Tongbian Capsules were prepared. The potential target proteins were captured from the total protein lysates of rat intestine using the beads. The captured proteins were further identified by LC-MS/MS, and the associated pathways were analyzed by Cytoscape. RESULTS:: showed that 138 potential target proteins were identified, which were involved in eight signaling pathways, including tricarboxylic acid cycle, pyrimidine metabolism, sulfur metabolism, fatty acid degradation, alanine/aspartate/glutamate metabolism, arginine/proline metabolism, valine/leucine/isoleucine degradation, and β-alanine metabolism. Taken together, Shouhui Tongbian Capsules may exert relaxing bowel effect by acting on multiple signaling pathways to promote intestinal gurgling, inhibit inflammation, as well as improve intestinal barrier function, intestinal water secretion, and intestinal flora.
Animals
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Capsules
;
Chromatography, Liquid
;
Intestines
;
Leucine
;
Rats
;
Tandem Mass Spectrometry
3.A 1-year-old female with maple syrup urine disease presenting with acrodermatitis-enteropathica-like lesions.
Rona Maria R. ABAD ; Johanna Pauline H. LAZO-DIZON
Acta Medica Philippina 2018;52(61):575-580
A 1-year-old female with maple syrup urine disease presenting with erythematous, partially eroded plaques on the trunk, anogenital area, and extremities experienced metabolic crisis. The skin lesions appeared at 11 months of age and was thought to result from amino acid imbalance secondary to erratic supplementation of specialized milk formula devoid of isoleucine, leucine, and valine. Serial urine monitoring showed persistent ketones and elevated serum leucine and valine. The patient was managed with emollients, intralipid 20%, and addition of isoleucine and valine supplements to counter the neurotoxic effect of leucine. After 8 days of proper feeding and continuous emollient application, the lesions improved and skin biopsy revealed superficial perivascular dermatitis. Although a decrease in erythema and desquamation was noted, the patient had persistent cerebral edema and continued to deteriorate.
Maple Syrup Urine Disease ; Isoleucine ; Leucine ; Valine ; Erythema
4.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
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Escherichia coli/genetics*
;
Leucine Dehydrogenase/genetics*
;
Threonine Dehydratase
5.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
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Cell Line, Tumor
;
Glioblastoma/metabolism*
;
Leucine
;
Leucine-Rich Repeat Proteins/genetics*
;
MicroRNAs
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Nerve Tissue Proteins/genetics*
;
Tumor Microenvironment
6.Detection of point mutation at C-terminal region of phagosomal coat protein (TACO) in patients with leprosy.
Se Kon KIM ; Tae Jin KANG ; Byoung Chul KIM ; Gue Tae CHAE
Korean Leprosy Bulletin 2003;36(1):11-26
Mycobacteria, which are highly successful pathogen, resist delivary to lysosomes and instead survive within a specialized vacuole, the mycobacterial phagosome. The bacteria survive intracellularly because they are able to actively recruit and retain TACO ( tryptophane aspartate-containing coat protein ) at the mycobacterial phagosome, where it prevents lysosomal delivary in a cholesterol-dependent manner. In this study, we investigated the difference of TACO expression is whether related to mutant in coro1a gene in patients with leprosy and normal volunteer. First, we screened for detection of a mutant in the leucine zipper motif within the exon 11, and then in the exon 9 to 10, and finally in the coiled-coil region. Interestingly, single base substitutions ( point mutation ) presents at assembly site of U1 snRNP, around of 5' splice site in the intron 9, there are a C to T and G to A transition are at 9 bp and 14 bp downstream of 5' splice site, respectively, and both of it. Among the 3 types of polymorphism, frequency of a G to A transition is markedly increased in patients of lepromatous type, which are new cases or relapsed. Both a C to T and G to A transitions are found in 1 case of tuberculoid type and 2 cases in lepromatoue type, but not found in control group. The silent mutation in leucine zipper motif within the exon 11 is located at codon at 454 ( CTG-->CTA), which is 1st leucine from C-terminal among four leucine zipper. In coiled-coil region, no mutation is found in genomic DNA of patients with leprosy. Further, we will do functional study about the identified point mutation and will screen any possible mutation in the region of promotor and WD repeat.
Bacteria
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Codon
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DNA
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Exons
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Healthy Volunteers
;
Humans
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Introns
;
Leprosy*
;
Leucine
;
Leucine Zippers
;
Lysosomes
;
Phagosomes
;
Point Mutation*
;
Ribonucleoprotein, U1 Small Nuclear
;
RNA Splice Sites
;
Tryptophan
;
Vacuoles
7.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
;
Electroencephalography
;
Leucine/genetics*
;
Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/genetics*
;
Mice
;
Mice, Transgenic
;
Mutation
;
Parkinson Disease/metabolism*
;
Sleep Deprivation/complications*
;
alpha-Synuclein/genetics*
8.Identification of Human LRG1 Polymorphisms and Their Genetic Association with Rheumatoid Arthritis.
Eun Heui JIN ; Soo Cheon CHAE ; Seung Cheol SHIM ; Hwan Gyu KIM ; Hun Taeg CHUNG
Genomics & Informatics 2008;6(2):77-83
Human leucine-rich alpha-2-glycoprotein 1 (LRG1 ) was first identified as a trace protein in human serum. The primary sequence of LRG1 includes repeated leucine residues and putative membrane-binding domains. But, there is no published information on the genetic variation of this gene. In this study, LRG1 was identified as one of several upregulated genes in RA patients. We examined the expression levels of LRG1 between an RA patient and a healthy control by RT-PCR and validated that LRG1 was highly expressed in RA patients compared with controls. We identified the possible variation sites and single nucleotide polymorphisms (SNPs) in the human LRG1 gene by direct sequencing and analyzed the association of genotype and allele frequencies between RA patients and a control group without RA. We further investigated the relationship between these polymorphisms and the level of RF or anti-CCP in RA patients. We identified a total of three SNPs (g.-678A> G, g.-404C>T and g.1427T>C) and two variation sites (g.-1198delA and g.-893delA) in the LRG1 gene. Our results suggest that polymorphisms of the LRG1 gene are not associated with the susceptibility of RA in the Korean population.
Arthritis, Rheumatoid
;
Gene Frequency
;
Genetic Variation
;
Genotype
;
Humans
;
Leucine
;
Polymorphism, Single Nucleotide
9.Relation among Calcium Intake, Bone Metabolism Parameters, Serum Protein and Lipids of Female College Students in Chungnam.
Mi Kyeong CHOI ; Chung Ja SUNG ; Mi Hyun KIM
Journal of the Korean Dietetic Association 2000;6(2):108-116
The purpose of this study was to investigate the relationship among calcium intake, blood parameters related with bone metabolism, and serum lipids in healthy adults on self-selected diet. Subjects were consisted of 40 female college students residing in Chungnam. Anthropometric measurements, dietary intake measurements and blood collection were conducted. Serum concentrations of total protein, albumin, alkaline phosphates, leucine amino peptidase, BUN, calcium, inorganic phosphorus, and lipids were measured by biochemical analyzer and ICP spectrometer. The results were as follows. The mean age of subjects was 22.34 years and weight, height and BMI were 52.89kg, 161.29cm and 20.34, respectively. The daily mean energy and calcium intakes were 81.75% and 64.38% of RDA. The mean animal 1:2. The mean serum concentrations were 6.54g/dl(total protein), 4.12g/dl(albumin), 123.24U/iota(alkaline phosphates), 36.59U/iota(leucine amino peptidase), 8.26mg/dl(calcium), 3.29mg/dl(inorganic phosphorus), 60.73mg/dl(triglyceride), 138.49mg/dl(total cholesterol), 65.95mg/dl(HDL-cholesterol), and 60.39mg/dl(LDL-cholesterol). There were no significant differences among calcium intake, bone metabolism parameters, and serum lipids when analyzed by Pearson's correlation coefficient. More systematic studies are required to investigate the roles of calcium in healthy persons on self-selected diets containing different levels of calcium.
Adult
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Animals
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Calcium*
;
Chungcheongnam-do*
;
Diet
;
Female*
;
Humans
;
Leucine
;
Metabolism*
;
Phosphates
;
Phosphorus
10.A Case of Lennox-Gastaut Syndrome due to 3-Methylcrotonyl CoA Carboxylase Deficiency.
Yu Sok HAN ; Hoon Chul KANG ; Hong Jin LEE ; Heung Dong KIM
Journal of the Korean Child Neurology Society 2004;12(1):92-98
3-Methylcrotonyl-CoA carboxylase(MCC) is a biotin-dependent enzyme involved in the leucine metabolism. We describe a patient with MCC deficiency who manifested with Reye syndrome-like illness with status epilepticus, metabolic acidosis, hypoglycemia, hyperammonemia, elevated liver enzymes and neurologic impairments after a viral gastroenteritis and then suffered from Lennox-Gastaut syndrome. Urinary organic acid analysis revealed increased excretions of 3-hydroxyisovaleric acid and 3-methylcrotonylglycine. This patient was managed with a leucine restriction diet and supplementation of biotin and carnitine, which was not so effective. He suffered from neurologic sequelae such as Lennox-Gastaut syndrome, motor and cognitive impairements.
Acidosis
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Biotin
;
Carnitine
;
Diet
;
Gastroenteritis
;
Humans
;
Hyperammonemia
;
Hypoglycemia
;
Leucine
;
Liver
;
Metabolism
;
Status Epilepticus