Reliable method for high quality His-tagged and untagged E. coli phosphoribosyl phosphate synthase (Prs) purification.

Prs (phosphoribosyl pyrophosphate synthase) is a broadly conserved protein that synthesises 5-phosphoribosyl 1-pyrophospate (PRPP); a substrate for biosynthesis of at least 10 enzymatic pathways including biosynthesis of DNA building blocks - purines and pyrimidines. In Escherichia coli, it is a protein of homo-hexameric quaternary structure, which can be challenging to work with, due to frequent aggregation and activity loss. Several studies showed brief purification protocols for various bacterial PRPP synthases, in most cases involving ammonium sulfate precipitation. Here, we provide a protocol for expression of E. coli Prs protein in Rosetta (DE3) and BL21 (DE3) pLysE strains and a detailed method for His-Prs and untagged Prs purification on nickel affinity chromatography columns. This protocol allows purification of proteins with high yield, purity and activity. We report here N-terminally His-tagged protein fusions, stable and active, providing that the temperature around 20 °C is maintained at all stages, including centrifugation. Moreover, we successfully applied this method to purify two enzyme variants with K194A and G9S alterations. The K194A mutation in conserved lysine residue results in protein variant unable to synthetize PRPP, while the G9S alteration originates from prs-2 allele variant which was previously related to thermo-sensitive growth. His-PrsG9S protein purified here, exhibited comparable activity as previously observed in-vivo suggesting the proteins purified with our protocol resemble their physiological state. The protocol for Prs purification showed here indicates guidance to improve stability and quality of the protein and to ensure more reliable results in further assays in-vitro.

Identification of high-risk stage II colorectal tumors by combined analysis of the NDRG1 gene expression and the depth of tumor invasion.

BACKGROUND: Experiments on cancer cell lines and animal models indicated that alteration in expression of N-myc down-regulated gene 1 (NDRG1) is associated with development of colon cancer. However, few clinical data are available to assess the role of NDRG1 in progression of human colorectal cancer. This study was undertaken to reveal the prognostic and predictive usefulness of NDRG1 expression determination in colorectal cancer. METHODS: The expression of NDRG1 mRNA was investigated in 108 colorectal cancer tissues by real-time polymerase chain reaction. The level of NDRG1 protein was investigated by immunohistochemistry. RESULTS: Patients with lowered level of NDRG1 mRNA had a statistically significantly shorter 5-year survival rate compared with patients with unchanged expression of NDRG1 (P = .01). The overall survival time for patients with II tumor, node, metastasis system (TNM) stage disease and tumors displaying reduced expression of NDRG1 was significantly shorter compared with patients with preserved NDRG1 expression (P = .024). Moreover, the survival rate of patients with TNM stage II disease and T4 lesion was significantly lower (P = .0005) for patients with reduced level of NDRG1 expression compared with patients with unchanged NDRG1 expression. The stepwise multivariate regression analysis revealed that advanced TNM stage and lowered NDRG1 expression level were independent unfavorable prognostic factors for patient survival. CONCLUSIONS: The assessment of NDRG1 expression offers valuable prognostic information for patients with colorectal cancer, especially for those with stage II disease. We propose that NDRG1 expression level could be used to select patients with stage II disease who are at increased risk of unfavorable outcome, and who may benefit from adjuvant therapy.

Different signaling pathways utilized by insulin to regulate the expression of ENT2, CNT1, CNT2 nucleoside transporters in rat cardiac fibroblasts.

In cardiac fibroblasts (CFs), insulin was shown to affect the expression of ENT2, CNT1, and CNT2 transporter. In the present study, we determined the signaling pathways utilized by insulin to regulate the expression of these nucleoside transporters. In the primary culture of rat CFs, insulin increased the mRNA level of ENT2 and suppressed the CNT1 and CNT2 mRNA levels. The insulin-induced increase of the ENT2 mRNA level was blocked by rapamycin (an inhibitor of mTOR) and by cycloheximide (an inhibitor of protein synthesis), whereas neither wortmannin (an inhibitor of PI3K) nor PD98059 (an inhibitor of MEK) affected the insulin action on the ENT2 transcript level. PD98059 completely blocked the insulin-induced decrease of the CNT1 and CNT2 mRNAs levels. Wortmannin prevented the insulin-induced change of the CNT1 mRNA level, but had no effect on the CNT2 mRNA. Rapamycin abolished the insulin effect on the CNT1 mRNA level, but not on the CNT2 mRNA. Cycloheximide prevented the insulin-induced decrease of CNT2 mRNA, but had no effect on the CNT1 mRNA level. Overall, our results demonstrate that the expression level of ENT2, CNT1, and CNT2 transporters in CFs is differentially regulated by insulin. Moreover, in this cell type insulin employs a distinct signaling pathway to regulate the expression of each transporter.

Ubc9-induced inhibition of diadenosine triphosphate hydrolase activity of the putative tumor suppressor protein Fhit.

Fhit protein is the product of the putative tumor suppressor fragile histidine triad (FHIT) gene. The way by which Fhit exerts its antitumor activity remains largely unknown, although the Fhit-Ap3A complex is believed to be the native signaling form of Fhit. Here, we have shown that Fhit protein interacts with hUbc9, a recombinant human SUMO-1 conjugating enzyme, in an adenosine(5')triphospho(5')nucleoside (Ap3N)-dependent manner. Our experiments showed that the dinucleoside polyphosphate hydrolase activity of Fhit is suppressed by interacting with hUbc9 protein. In the presence of equimolar hUbc9 the Vmax and Km activity of Fhit was decreased by 35%. Analysis of Fhit kinetics in the presence of different fixed concentrations of Ubc9 showed that Ubc9 is an uncompetitive inhibitor. Including SUMO-1 protein in the assay neither affected the Fhit activity nor modified the effect of Ubc9 on Fhit kinetics. Our data suggest that hUbc9-induced inhibition of Fhit may result in an elongation of the Fhit-Ap3A signaling complex lifetime leading to alteration of its antitumor activity.

Expression level of Ubc9 protein in rat tissues.

Ubc9 is a homologue of the E2 ubiquitin conjugating enzyme and participates in the covalent linking of SUMO-1 molecule to the target protein. In this report we describe a simple and efficient method for obtaining pure human recombinant Ubc9 protein. The purified Ubc9 retained its native structure and was fully active in an in vitro sumoylation assay with the promyelocytic leukaemia (PML) peptide as a substrate. In order to better understand the physiology of Ubc9 protein we examined its levels in several rat tissues. Immunoblot analyses performed on tissue extracts revealed quantitative and qualitative differences in the expression pattern of Ubc9. The Ubc9 protein was present at a high level in spleen and lung. Moderate level of Ubc9 was detected in kidney and liver. Low amount of Ubc9 was observed in brain, whereas the 18 kDa band of Ubc9 was barely visible or absent in heart and skeletal muscle. In heart and muscle extracts the Ubc9 antibodies recognized a 38 kDa protein band. This band was not visible in extracts of other rat tissues. A comparison of the relative levels of Ubc9 mRNA and protein indicated that the overall expression level of Ubc9 was the highest in spleen and lung. In spleen, lung, kidney, brain, liver and heart there was a good correlation between the 18 kDa protein and Ubc9 mRNA levels. In skeletal muscle the Ubc9 mRNA level was unproportionally high comparing to the level of the 18 kDa protein. The presented data indicate that in the rat the expression of the Ubc9 protein appears to have some degree of tissue specificity.