[Show abstract][Hide abstract] ABSTRACT: Human Bex2 (brain expressed X-linked, hBex2) is highly expressed in the embryonic brain, but its function remains unknown. We have identified that LMO2, a LIM-domain containing transcriptional factor, specifically interacts with hBex2 but not with mouse Bex1 and Bex2. The interaction was confirmed both by pull-down with GST-hBex2 and by coimmunoprecipitation assays in vivo. Using electrophoretic mobility shift assay, we have demonstrated the physical interaction of hBex2 and LMO2 as part of a DNA-binding protein complex. We have also shown that hBex2 can enhance the transcriptional activity of LMO2 in vivo. Furthermore, using mammalian two-hybrid analysis, we have identified a neuronal bHLH protein, NSCL2, as a novel binding partner for LMO2. We then showed that LMO2 could up-regulate NSCL2-dependent transcriptional activity, and hBex2 augmented this effect. Thus, hBex2 may act as a specific regulator during embryonic development by modulating the transcriptional activity of a novel E-box sequence-binding complex that contains hBex2, LMO2, NSCL2 and LDB1.
Nucleic Acids Research 02/2005; 33(20):6555-65. · 8.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Nervous system polycomb 1 (NSPc1) shares high homology with vertebrate PcG proteins Mel-18 and Bmi-1. The mRNA of NSPc1 is highly expressed in the developmental nervous system [Mech. Dev. 102 (2001) 219-222]. However, the functional characterization of NSPc1 protein is not clear. In the present study, using Western blotting technique, we aimed to describe the distributions of NSPc1 protein in rat tissues and cell lines. The subcellular localization of NSPc1 was examined in HeLa and SH-SY5Ycell lines, and its transcriptional repression activity was examined in COS-7 cell line. We found that the NSPc1 protein was localized mainly in the nucleus. NSPc1 remarkably repressed the transcription. Most interestingly, both the C-terminal of NSPc1 and two phosphorylation sites in the C-terminal, especially the PKC phosphorylation site at S183, were important in mediating transcription repression. Taken together, results from our study suggest that NSPc1, as a typical PcG family member, has powerful transcriptional repression ability, which may be related to the PKC signaling pathway.
[Show abstract][Hide abstract] ABSTRACT: Proteins of the immunoglobulin superfamily (IgSF) are involved in a variety of specific cell-cell interactions in the developing nervous system. To identify and characterize new members of this protein family in human nervous system, we screen the human fetal brain cDNA library and isolate a full-length cDNA clone which contains a 1032 bp open reading frame encoding a protein of 344 amino acids. Sequence analysis reveals that it is a glycoprotein comprised of three C2-like immunoglobulin domains and is anchored to the plasma membrane via a post-translationally attached glycosyl-phosphatidylinositol (GPI) moiety. The protein shows high sequence similarity to the rat Ntm (97%), so we term it human neurotrimin (NTM). Northern blot analysis reveals that (HUMAN)NTM has three different transcripts with the length of 3.2 kb, 4.0 kb and 9.0 kb respectively. It has a wider expression pattern than that of (RAT) Ntm. Notably, the expression of NTM in fetal brain is higher than that in mature brain and is stronger in nervous tumors than that in normal brain tissues. We insert an HA epitope tag between the third Ig-like domain of NTM and the site of GPI attachment, then construct it into the eukaryotic expression vector pcDNA3.1+/Zeocin. The pcDNA3.1-HA-NTM is transfected into the Chinese Hamster Ovary (CHO) cells. The results demonstrate that HA-NTM is expressed on the surface of CHO cells and could strengthen the aggregation of CHO-NTM cells.
Science in China Series C Life Sciences 05/2004; 47(2):158-64. · 1.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The PTB domain of mouse dok1 fusion protein has been overexpressed in Escherichia coli and crystallized in a form suitable for X-ray crystallographic study. Crystals have been obtained using the vapour-diffusion method and belong to space group P2(1)2(1)2(1). X-ray diffraction data were collected in-house to 2.5 A resolution. A selenomethionine (SeMet) dok1 PTB fusion-protein derivative was expressed using the same expression system, purified in a reductive environment and crystals were obtained under similar conditions. Subsequently, three different wavelength data sets from the derivative crystal were collected to 2.5 A resolution at SPring-8.
[Show abstract][Hide abstract] ABSTRACT: Dok1 is a common substrate of activated protein-tyrosine kinases. It is rapidly tyrosine-phosphorylated in response to receptor tyrosine activation and interacts with ras GTPase-activating protein and Nck, leading to inhibition of ras signaling pathway activation and the c-Jun N-terminal kinase (JNK) and c-Jun activation, respectively. In chronic myelogenous leukemia cells, it has shown constitutive phosphorylation. The N-terminal phosphotyrosine binding (PTB) domain of Dok1 can recognize and bind specifically to phosphotyrosine-containing motifs of receptors. Here we report the crystal structure of the Dok1 PTB domain alone and in complex with a phosphopeptide derived from RET receptor tyrosine kinase. The structure consists of a beta-sandwich composed of two nearly orthogonal, 7-stranded, antiparallel beta-sheets, and it is capped at one side by a C-terminal alpha-helix. The RET phosphopeptide binds to Dok1 via a surface groove formed between strand beta5 and the C-terminal alpha-helix of the PTB domain. The structures reveal the molecular basis for the specific recognition of RET by the Dok1 PTB domain. We also show that Dok1 does not recognize peptide sequences from TrkA and IL-4, which are recognized by Shc and IRS1, respectively.
Journal of Biological Chemistry 03/2004; 279(6):4962-9. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Until 30 years ago, it had been considered that D-amino acids were excluded from living systems except for D-amino acids in the cell wall of microorganisms. However, D-amino acids, in the form of free amino acids, peptides and proteins, were recently detected in various living organisms from bacteria to mammals. The extensive distribution of bio-functional D-amino acids challenges the current concept of protein synthesis: more attention should be paid to the stereospecificity of the translation machine. Besides aminoacyl-tRNA synthetases, elongation factor Tu and some other mechanisms, D-Tyr-tRNA(Tyr) deacylases provide a novel checkpoint since they specifically recycle misaminoacylated D-Tyr-tRNA(Tyr) and some other D-aminoacyl-tRNAs. Their unique structure represents a new class of tRNA-dependent hydrolase. These unexpected findings have far-reaching implications for our understanding of protein synthesis and its origin.
[Show abstract][Hide abstract] ABSTRACT: For better understanding of functions of the Calcyclin Binding Protein (CacyBP) and exploring its possible roles in neuronal differentiation, the subcellular localization of human CacyBP was examined in retinoic acid(RA)-induced and uninduced neuroblastoma SH-SY5Y cells. Immunostaining indicated that CacyBP was present in the cytoplasm of uninduced SH-SY5Y cells, in which the resting Ca(2+) concentration was relatively lower than that of RA-induced cells. After the RA induction, immunostaining was seen in both the nucleus and cytoplasm. In the RA-induced differentiated SH-SY5Y cells, CacyBP was phosphorylated on serine residue(s), while it existed in a dephosphorylated form in normal (uninduced) cells. Thus, the phosphorylation of CacyBP occurs when it is translocated to the nuclear region. The translocation of CacyBP during the RA-induced differentiation of SH-SY5Y cells suggested that this protein might play a role in neuronal differentiation.
Journal of biochemistry and molecular biology 08/2003; 36(4):354-8. · 2.02 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The human dok-5 PTB domain fusion protein has been overexpressed in Escherichia coli and crystallized in a form suitable for X-ray crystallographic study. Crystals were obtained by the vapour-diffusion method. The crystal has unit-cell parameters a = b = 75.9, c = 108.0 A, alpha = beta = 90, gamma = 120 degrees and belongs to space group P3(2)21. Diffraction data were collected to 2.8 A resolution in-house. Furthermore, a selenomethionine (SeMet) derivative of dok-5 PTB domain fusion protein was overexpressed using the same expression system and was purified in a reductive environment. The derivative crystals were obtained under similar conditions. Subsequently, three different wavelength data sets were collected to 2.3 A resolution from the derivative crystal at the Advanced Photon Source, Argonne National Laboratory.
[Show abstract][Hide abstract] ABSTRACT: Thioredoxin is a ubiquitous dithiol oxidoreductase found in many organisms and involved in numerous biochemical processes. Human thioredoxin-like protein (hTRXL) is differentially expressed at different development stages of human fetal cerebrum and belongs to an expanding family of thioredoxins. We have solved the crystal structure of the recombinant N-terminal catalytic domain (hTRXL-N) of hTRXL in its oxidized form at 2.2-A resolution. Although this domain shares a similar three-dimensional structure with human thioredoxin (hTRX), a unique feature of hTRXL-N is the large number of positively charged residues distributed around the active site, which has been implicated in substrate specificity. Furthermore, the hTRXL-N crystal structure is monomeric while hTRX is dimeric in its four crystal structures (reduced, oxidized, C73S and C32S/C35S mutants) reported to date. As dimerization is the key regulatory factor in hTRX, the positive charge and lack of dimer formation of hTRXL-N suggest that it could interact with the acidic amino-acid rich C-terminal region, thereby suggesting a novel regulation mechanism.
European Journal of Biochemistry 05/2002; 269(8):2060-8. · 3.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To clone and identify the gene encoding human ubiquitin binding enzyme 2 and study its expression pattern.
According to the sequence of human EST, which is highly homologous to the mouse ubiquitin binding/conjugating enzyme (E2), primers were synthesized to screen the human fetal brain cDNA library. The gene was analyzed by bioinformatics technique and its expression pattern was studied by using multiple-tissue Northern blot.
Two cDNA clones encoding human ubiquitin conjugating enzyme have been isolated and identified. Both containing the ubiquitin conjugating domain, the 2 cDNA clones are 88% identical in amino acid sequences and splicing isoforms to each other only with an exon excised to form the short sequence. They belong to a highly conserved and widely expressed E2 enzyme family. Northern blot shows that they are expressed exclusively in adult human heart, placenta, and pancreas but no transcripts can be detected in brain, lung, liver, skeletal muscle or kidney.
The gene encoding human ubiquitin binding enzyme is expressed under temporal control. As a key enzyme in the degradation of proteins, ubiquitin conjugating enzymes play a central role in the expression regulation on the level of post-translation.
Chinese Medical Sciences Journal 04/2002; 17(1):7-12.
[Show abstract][Hide abstract] ABSTRACT: LHX4 gene is a member of the LIM-homeobox gene family and plays a critical role in the development of motor neurons. We have isolated a cDNA of human LHX4 from a library of the adult human spinal cord. Its sequence is 92% homologous to that of the mouse Lhx4. The genomic structure of the LHX4 gene and its chromosomal localization were determined. The gene was mapped on human chromosome 1q 24.1-1q 24.3 and composed of six exons. The homeodomain was encoded by two exons, exons 4 and 5. The first LIM domain was coded by exon 2, and the second by exon 3. Human MTE Array was used to study the expression profile of LHX4 in 72 human tissues. The expression was specific in the CNS including the fetal brain, the spinal cord, and the cerebral cortex. In situ hybridization of the adult rodent CNS showed the abundant expression of LHX4 in the cerebral cortex and motor neurons of the spinal cord. Our results suggest that LHX4 may play a role in the CNS, especially the neocortex and the spinal cord, and provide a basis to investigate potential involvement of the LHX4 gene in human diseases.
Brain Research 03/2002; 928(1-2):147-55. · 2.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In order to identify the genes associated with glioblastoma differentiation, some ESTs, expressed differentially in the control cell and the differentiated human glioblastoma cell line BT-325 induced by the all-trans retinoid acid, have been isolated by the method of DDRT-PCR. Of the 46 ESTs sequenced, 19 are from new genes. A full-length 1 535-bp cDNA, termed geneGDR1, has been isolated from the human cDNA library using the probe designed according to one of the novel ESTs, HGBB098. The open reading frame ofGDR1 gene encodes a putative protein containing 334 amino acid residues. Blast against the current GenBank DNA and protein sequence database did not reveal significant homology with any known proteins. RT-PCR shows thatGDR1 mRNA level increased in the differentiated BT-325 cells after being treated with RA. The different expression patterns ofGDR1 mRNA in human tissues have been detected through the multiple tissue Northern blot hybridization.
Chinese Science Bulletin 06/2000; 45(11):995-999. · 1.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Thembl (muscleblind) gene ofDrosophila encodes a nuclear protein which contains two Cys3His motifs. The mutation ofmbl gene will disturb the differentiation of all theDrosophila’s photoreceptors. Primers have been designed according to human EST086139, which is highly homologous tombl gene. Human fetal brain cDNA library has been screened and a novel cDNA clone has been obtained. The 2595 bp cDNA, designatedMBLL (muscleblind-like), contains an open reading frame which encodes 255 amino acids and has 4 Cys3His motifs (GenBank Acc. AF061261). The amino acids sequence shares high homology toDrosophila’s mbl. The Northern blot and RNA dot blot hybridization of 43 human adult tissues and 7 fetal tissues show thatMBLL is a widely expressed gene, but the expression amounts differ in these tissues.
Chinese Science Bulletin 03/2000; 45(7):620-625. · 1.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Copper is one of the most important trace elements to life. HumanHCUTA is a novel cDNA encoding a 156aa protein, which may participate in human copper tolerance system. The HCUTA protein is highly
similar to protein CUT A1 ofE. coli. The whole opening reading frame ofHCUTA cDNA was amplified by PCR and cloned into pET28a + express vector, and the HCUTA protein was effectively expressed inE. coli BL21 (DE3).
Chinese Science Bulletin 01/2000; 45(14):1301-1304. · 1.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: After screening human fetal brain cDNA library, a novel human cDNA encoding an RNA-binding protein was cloned and namedGRY-RBP. HumanGRY-RBP cDNA is 2 932 bp in length, and encodes a protein of 623 amino acids with a deduced molecular mass of 69.6 ku. Human GRY-RBP contains three RNA recognition motifs (RRMs) and a glycine (G), tyrosin (Y)-rich C-terminus. Computer searches with sequence database revealed its moderate homology with RRMs of many RRM RNA-binding proteins. In this study, the full coding region and partial non-coding region of mouseGRY-RBP cDNA were amplified by PCR using mouse brain cDNA library as template. Three transcripts ofGry-rbp are expressed in all tissues as shown by Northern blot, but the amounts in heart, planceta and skeletal muscle are considerably higher than those in other tissues.
Chinese Science Bulletin 01/2000; 45(4). · 1.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: mRNA differential display (DDRT-PCR) has been used to analyze different human fetal brain tissues of different developmental
stages (13- and 33-week). According to the sequence of one EST obtained in this assay, a pair of primers have been designed
to screen the arrayed human fetal brain cDNA library. A 1.2-kb cDNA clone has been found. This cDNA consists of an 867 bp
open reading frame, a 132 bp 5′ untranslated sequence and a 209 bp 3′ untranslated sequence with a typical polyadenylation
signal. The coding region predicts a protein of 289 amino acids. Its N-terminal of 105 residues is highly homologous to human
thioredoxin, while no homology has been found in the databases with its C-terminal of 184 residues. Its N-terminal region
also contains the conserved active site sequence CGPC (Cys-Gly-Pro-Cys) of thioredoxin. It was named human Thioredoxin-like
Chinese Science Bulletin 09/1999; 44(18):1673-1676. · 1.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Genes encoding proteins with PHD (plant homeodomain) finger motif (C4HC3) are highly conserved fromArabidopis toHomo sapiens. One of the major functions of these genes is regulating the expression of homeotic genes during the stage of embryonic development.
They play a role in cell-cycle and cell differentiation and seem to be related with some human malignant diseases, such as
leukemia. A human placenta cDNA library has been screened with cDNA probe amplified by PCR. The PCR primers have been designed
according to theM96 (a mouse gene encoding a protein with PHD domain) homologous data in dbEST. A 2.1 kb insert fragment in a positive cDNA clone
has been isolated and sequenced. This new full-length cDNA is namedPHF2 (GenBank Acc: AF052205). The putative protein composed of 567 aa has two typical PHD fingers at its N-terminus. Meanwhile
it is identified that there are several alternatively spliced transcripts ofPHF2 in different human tissues through the PCR amplification, Northern blot hybridization and analysis of genomic DNA structure.
Chinese Science Bulletin 07/1999; 44(15):1382-1387. · 1.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Primers for screening cDNA library have been designed according to EST AA453734 which is corresponding to the mouse LIM domain
binding protein Ldbl. Arrayed human fetal brain cDNA library has been screened by PCR and routine hybridization method. A
2398 bp-cD-NA clone has been obtained. The cDNA encodes a 347 amino acids protein highly homologous to the mouse Ldbl,Xenopus Xldbl andDrosophila Chip. It also contains an LIM binding domain and a nuclear localization signal. It has been namedLDB1 ( LIM domain binding protein 1), GenBank accession number is AF052389. Northern blot showed a 2.4 kb band, and the expression
amounts ofLDBI in heart, brain and lung were considerably higher than those in other tissues.
Chinese Science Bulletin 01/1999; 44(12):1114-1119. · 1.37 Impact Factor