Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.2.1.23 (beta-galactosidase)
 14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Smac/DIABLO (second mitochondria-derived activator of caspase/direct IAP-binding protein with low PI) is a 29 kDa mitochondrial precursor protein, which is proteolytically processed in mitochondria into a 23 kDa mature protein. It is released from the mitochondrial intermembrane space to cytosol after an apoptotic trigger. Smac/DIABLO acts as a dimer and it contributes to caspase activation by sequestering the inhibitor of apoptosis proteins (IAPs). In order to further investigate the mechanism of Smac/DIABLO action, we used the mature form of Smac/DIABLO as a bait and screened proteins that interact with mature Smac/DIABLO in human liver cDNA library using the yeast two-hybrid system. Forty-two colonies were obtained after 5.8x10(6) colonies were screened by nutrition limitation and X-galactosidase assay. After DNA sequence analysis and homology retrieval, one of the candidate proteins was identified as TRAF domain of the TNF receptor associated factor 3 (TRAF3). The interaction site between TRAF3 and Smac/DIABLO was identified by beta-galactosidase test. The interaction between TRAF3 and Smac/DIABLO via TRAF domain was identified in vivo by co-immunoprecipitation in HepG2 cells, and the direct interaction between TRAF3 and Smac/DIABLO in vitro was identified by GST-pull down assay. Co-expression of TRAF3 and mature Smac/DIABLO in 293 cells could enhance the Smac/DIABLO-mediated apoptosis. These results suggested that TRAF3 interacted with Smac/DIABLO via TRAF domain, leading to an increased proapoptotic effect of Smac/DIABLO in cytoplasm.
 ...
PMID:TRAF3 interacts with Smac/DIABLO and enhances the proapoptotic effect of Smac/DIABLO in cytoplasm. 1727 85

pGEX vectors are widely used for GST-fusion protein expression in Escherichia coli under the control of a strong IPTG inducible tac promoter. While using pGEX-4T-2 vector in heterologous protein expression we noticed that the GST or GST-fusion protein were expressed at a very low levels. Interestingly, we found a spontaneous deletion of 701 bp DNA fragment harbouring the tac promoter in both, native and recombinant pGEX-4T-2 vectors. This deletion took place between two direct repeats of 43 bases and led to the loss of a 701 bp DNA fragment. This explained the decrease in GST or GST-fused protein level since the tac promoter, that directs transcription was deleted. The lacZ promoter, located upstream of the deleted fragment, replaced tac promoter but was less efficient. The deleted DNA also specifies part of the lacZ gene coding for the N-terminal end of the beta-galactosidase (the alpha-peptide), which is slightly functional. Consequently, bacterial cells transformed with the original pGEX are of a faint blue colour while those bearing the deleted ones are white, when plated on X-Gal containing medium. The deletion, did not affect neither the sequence nor the molecular weight of GST and fusion protein since it took place just before the GST start codon. It occurred in E. coli TOP10 cells which are deficient in RecA protein, suggesting that the deletion did not require the RecA recombination system.
 ...
PMID:A spontaneous direct repeat deletion in the pGEX fusion vector decreases the expression level of recombinant proteins in Escherichia coli. 1843 94

Polo-like kinase 1 plays an essential role in mitosis and cytokinesis. Expression and nuclear localization of Plk1 during the S phase are necessary for its functions. Although it was reported that a bipartite nuclear localization signal located at the N-terminal kinase domain is required for nuclear import of Plk1, Plk1 carrying mutations in the polo box I of the polo box domain exhibited increased cytoplasmic accumulation. We further showed that the polo box domain was able to confer nuclear import of beta-galactosidase in vivo and GST-EGFP in vitro. The import carriers transportin and importin alpha were found to interact with the polo box domain directly in a Ran-GTP sensitive manner. These results indicate the presence of a nuclear localization signal in the polo box domain. A 38 amino acid sequence with the function of nuclear localization signal was identified to interact with transportin. Our findings demonstrated that a transportin-dependent nuclear localization signal is present in the polo box domain of Plk1, possibly required for efficient nuclear import. Showing little similarity to the M9 sequence, the 38 amino acid sequence identified here likely represents a novel nuclear localization signal.
 ...
PMID:Identification of a nuclear localization signal in the polo box domain of Plk1. 1963 97

The strychnine-sensitive glycine receptor (GlyR) is a ligand-gated ion channel that mediates fast synaptic inhibition in the vertebrate central nervous system. As a member of the family of Cys-loop receptors, it assembles from five homologous subunits (GlyRalpha1-4 and -beta). Each subunit contains an extracellular ligand binding domain, four transmembrane domains (TM), and an intracellular domain, formed by the loop connecting TM3 and TM4 (TM3-4 loop). The TM3-4 loops of the subunits GlyRalpha1 and -alpha3 harbor a conserved basic motif, which is part of a potential nuclear localization signal. When tested for functionality by live cell imaging of green fluorescent protein and beta-galactosidase-tagged domain constructs, the TM3-4 loops of GlyRalpha1 and -alpha3, but not of GlyRalpha2 and -beta, exhibited nuclear sorting activity. Subunit specificity may be attributed to slight amino acid alterations in the basic motif. In yeast two-hybrid screening and GST pulldown assays, karyopherin alpha3 and alpha4 were found to interact with the TM3-4 loop, providing a molecular mechanism for the observed intracellular trafficking. These results indicate that the multifunctional basic motif of the TM3-4 loop is capable of mediating a karyopherin-dependent intracellular sorting of full-length GlyRs.
 ...
PMID:Multifunctional basic motif in the glycine receptor intracellular domain induces subunit-specific sorting. 1995 65

Expression and secretion of recombinant proteins in the endotoxin-free bacterium, Bacillus subtilis, has been thoroughly studied, but overexpression in the cytoplasm has been limited to only a few proteins. Here, we used the robust IPTG-inducible promoter, Pgrac212, to overexpress human rhinovirus 3C protease (HRV3C) in the cytoplasm of B. subtilis cells. A novel solubility tag, the N-terminal domain of the lysS gene of B. subtilis coding for a lysyl-tRNA synthetase was placed at the N terminus with a cleavage site for the endoprotease HRV3C, followed by His-HRV3C or His-GST-HRV3C. The recombinant protease was purified by using a Ni-NTA column. In this study, the His-HRV3C and His-GST-HRV3C proteases were overexpressed in the cytoplasm of B. subtilis at 11% and 16% of the total cellular proteins, respectively. The specific protease activities were 8065 U/mg for His-HRV3C and 3623 U/mg for His-GST-HRV3C. The purified enzymes were used to cleave two different substrates followed by purification of the two different protein targets, the green fluorescent protein and the beta-galactosidase. In conclusion, the combination of an inducible promoter Pgrac212 and a solubility tag allowed the overexpression of the HRV3C protease in the cytoplasm of B. subtilis. The resulting fusion protein was purified using a nickel column and was active in cleaving target proteins to remove the fusion tags. This study offers an effective method for producing recombinant proteins in the cytoplasm of endotoxin-free bacteria.
 ...
PMID:Using the IPTG-Inducible Pgrac212 Promoter for Overexpression of Human Rhinovirus 3C Protease Fusions in the Cytoplasm of Bacillus subtilis Cells. 3161 59


<< Previous 1 2