Leucine-rich repeats containing 4 protein (LRRC4) in memory, psychoneurosis, and glioblastoma / 中华医学杂志(英文版)

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.

Eukaryotic expression and antigen epitope prediction of the LRRC15 protein in excretory secretory antigens of Taenia solium cysticercus / 中国血吸虫病防治杂志

OBJECTIVE@#To conduct eukaryotic expression of the leucine-rich repeat containing 15 (LRRC15), a differentially expressed protein in excretory secretory antigens of Taenia solium cysticercus, and predict its antigen epitope.@*METHODS@#The molecular weight, stability, amino acid sequence composition, isoelectric point and T lymphocyte epitope of the LRRC15 protein were predicted using the bioinformatics online softwares ExPASy-PortParam and Protean. The full-length splicing primers were designed using PCR-based accurate synthesis, and the LRRC15 gene was synthesized. The recombinant pcDNA3.4-LRRC15 plasmid was constructed and transfected into HEK293 cells to express the LRRC15 protein. In addition, the LRRC15 protein was characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting.@*RESULTS@#The recombinant pcDNA3.4-LRRC15 plasmid was successfully constructed, which expressed the target LRRC15 protein with an approximately molecular weight of 70 kDa. Bioinformatics prediction with the ExPASy-PortParam software showed that LRRC15 was a hydrophilic protein, which was consisted of 644 amino acids and had a molecular weight of 69.89 kDa and an isoelectric point of 5.6. The molecular formula of the LRRC15 protein was C3073H4942N846O953S28 and had an instability coefficient is 50.3, indicating that LRRC15 was an instable protein. Bioinformatics prediction with the Protean software showed that the dominant T-cell antigen epitopes were located in 292 to 295, 353 to 361, 521 to 526 and 555 to 564 amino acids of the LRRC15 protein, and the T-cell antigen epitopes with a high hydrophilicity, good flexibility, high surface accessibility and high antigenicity index were found in 122 to 131, 216 to 233, 249 to 254, 333 to 343, 358 to 361, 368 to 372, 384 to 386, 407 to 412, 445 to 450, 469 to 481, 553 to 564, 588 to 594, 607 to 617 and 624 to 639 amino acids. Following transfection of the recombinant pcDNA3.4-LRRC15 plasmid into HEK293 cells, SDS-PAGE and Western blotting identified LRRC15 proteins in cell secretory culture media, cell lysis supernatants and sediments. The LRRC15-His fusion protein was purified from the cell culture medium, and SDS-PAGE identified a remarkable band at approximately 70 kDa, while Western blotting successfully recognized the band of the recombinant LRRC15 protein.@*CONCLUSIONS@#The eukaryotic expression and antigen epitope prediction of the LRRC15 protein in the excretory secretory antigens of T. solium cysticercus have been successfully performed, which provides insights into further understandings of its biological functions.

Recombinant Expression and Purification of F-box and Leucine-rich Repeat Protein 5 (FBXL5) using a Prokaryotic Expression System

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.