BMC Biochemistry最新文献

Background: Human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomesase, is responsible for telomere maintenance and its reactivation is implicated in almost 90% human cancers. Recent evidences show that hTERT is essential for neoplastic transformation independent of its canonical function. However, the roles of hTERT in the process remain elusive. In the current work, we explore the extra-telomeric role of hTERT in the neoplastic transformation of fibroblast IMR90.

Results: Here we established transformed IMR90 cells by co-expression of three oncogenic factors, namely, H-Ras, SV40 Large-T antigen and hTERT (RSH). The RSH-transformed cells acquired hallmarks of cancer, such as they can grow under anchorage independent conditions; self-sufficient in growth signals; attenuated response to apoptosis; and possessed recurrent chromosomal abnormalities. Furthermore, the RSH-transformed cells showed enhanced migration capability which was also observed in IMR90 cells expressing hTERT alone, indicating that hTERT plays a role in cell migration, and thus possibly contribute to their metastatic potential during tumor transformation. This notion was further supported by our microarray analysis. In addition, we found that Ku70 were exclusively upregulated in both RSH-transformed IMR90 cells and hTERT-overexpressing IMR90 cells, suggesting the potential role of hTERT in DNA damage response (DDR).

Conclusions: Collectively, our study revealed the extra-telomeric effects of hTERT in cell migration and DDR during neoplastic transformation.

Background: Cytochrome c (Cyt c) is an apoptosis-initiating protein when released into the cytoplasm of eukaryotic cells and therefore a possible cancer drug candidate. Although proteins have been increasingly important as pharmaceutical agents, their chemical and physical instability during production, storage, and delivery remains a problem. Chemical glycosylation has been devised as a method to increase protein stability and thus enhance their long-lasting bioavailability.

Results: Three different molecular weight glycans (lactose and two dextrans with 1 kD and 10 kD) were chemically coupled to surface exposed Cyt c lysine (Lys) residues using succinimidyl chemistry via amide bonds. Five neo-glycoconjugates were synthesized, Lac4-Cyt-c, Lac9-Cyt-c, Dex5(10kD)-Cyt-c, Dex8(10kD)-Cyt-c, and Dex3(1kD)-Cyt-c. Subsequently, we investigated glycoconjugate structure, activity, and stability. Circular dichroism (CD) spectra demonstrated that Cyt c glycosylation did not cause significant changes to the secondary structure, while high glycosylation levels caused some minor tertiary structure perturbations. Functionality of the Cyt c glycoconjugates was determined by performing cell-free caspase 3 and caspase 9 induction assays and by measuring the peroxidase-like pseudo enzyme activity. The glycoconjugates showed ≥94% residual enzyme activity and 86 ± 3 to 95 ± 1% relative caspase 3 activation compared to non-modified Cyt c. Caspase 9 activation by the glycoconjugates was with 92 ± 7% to 96 ± 4% within the error the same as the caspase 3 activation. There were no major changes in Cyt c activity upon glycosylation. Incubation of Dex3(1 kD)-Cyt c with mercaptoethanol caused significant loss in the tertiary structure and a drop in caspase 3 and 9 activation to only 24 ± 8% and 26 ± 6%, respectively. This demonstrates that tertiary structure intactness of Cyt c was essential for apoptosis induction. Furthermore, glycosylation protected Cyt c from detrimental effects by some stresses (i.e., elevated temperature and humidity) and from proteolytic degradation. In addition, non-modified Cyt c was more susceptible to denaturation by a water-organic solvent interface than its glycoconjugates, important for the formulation in polymers.

Conclusion: The results demonstrate that chemical glycosylation is a potentially valuable method to increase Cyt c stability during formulation and storage and potentially during its application after administration.

Background: Urease, one of the highly efficient known enzymes, catalyzes the hydrolysis of urea into ammonia and carbon dioxide. The present study aimed to extract urease from pea seeds (Pisum Sativum L). The enzyme was then purified in three consequence steps: acetone precipitation, DEAE-cellulose ion-exchange chromatography, and gel filtration chromatography (Sephacryl S-200 column).

Results: The purification fold was 12.85 with a yield of 40%. The molecular weight of the isolated urease was estimated by chromatography to be 269,000 Daltons. Maximum urease activity (190 U/g) was achieved at the optimum conditions of 40°C and pH of 7.5 after 5 min of incubation. The kinetic parameters, Km and Vmax, were estimated by Lineweaver-Burk fits and found to be 500 mM and 333.3 U/g, respectively. The thermodynamic constants of activation, ΔH, Ea, and ΔS, were determined using Arrhenius plot and found to be 21.20 kJ/mol, 23.7 kJ/mol, and 1.18 kJ/mol/K, respectively.

Conclusions: Urease was purified from germinating Pisum Sativum L. seeds. The purification fold, yield, and molecular weight were determined. The effects of pH, concentration of enzyme, temperature, concentration of substrate, and storage period on urease activity were examined. This may provide an insight on the various aspects of the property of the enzyme. The significance of extracting urease from different sources could play a good role in understanding the metabolism of urea in plants.

Background: Secreted luciferases are highly useful bioluminescent reporters for cell-based assays and drug discovery. A variety of secreted luciferases from marine organisms have been described that harbor an N-terminal signal peptide for release along the classical secretory pathway. Here, we have characterized the secretion of Gaussia luciferase in more detail.

Results: We describe three basic mechanisms by which GLUC can be released from cells: first, classical secretion by virtue of the N-terminal signal peptide; second, internal signal peptide-mediated secretion and third, non-conventional secretion in the absence of an N-terminal signal peptide. Non-conventional release of dNGLUC is not stress-induced, does not require autophagy and can be enhanced by growth factor stimulation. Furthermore, we have identified the golgi-associated, gamma adaptin ear containing, ARF binding protein 1 (GGA1) as a suppressor of release of dNGLUC.

Conclusions: Due to its secretion via multiple secretion pathways GLUC can find multiple applications as a research tool to study classical and non-conventional secretion. As GLUC can also be released from a reporter construct by internal signal peptide-mediated secretion it can be incorporated in a novel bicistronic secretion system.

Background: During EGFR internalization CIN85 bridges EGFR-Cbl complex, endocytic machinery and fusible membrane through the interactions of CIN85 with c-Cbl, endophilins and phosphatidic acid. These protein-protein and protein-lipid interactions are mediated or regulated by the positively charged C-terminal coiled-coil domain of CIN85. However, the details of CIN85-lipid interaction remain unknown. The present study suggested a possible electric interaction between the negative charge of phosphatidic acid and the positive charge of basic amino acids in coiled-coil domain.

Results: Mutations of the basic amino acids in the coiled-coil domain, especially K645, K646, R648 and R650, into neutral amino acid alanine completely blocked the interaction of CIN85 with c-Cbl or phosphatidic acid. However, they did not affect CIN85-endophilin interaction. In addition, CIN85 was found to associate with the internalized EGFR endosomes. It interacted with several ESCRT (Endosomal Sorting Complex Required for Transport) component proteins for ESCRT assembly on endosomal membrane. Mutations in the coiled-coil domain (deletion of the coiled-coil domain or point mutations of the basic amino acids) dissociated CIN85 from endosomes. These mutants bound the ESCRT components in cytoplasm to prevent them from assembly on endosomal membrane and inhibited EGFR sorting for degradation.

Conclusions: As an adaptor protein, CIN85 interacts with variety of partners through several domains. The positive charges of basic amino acids in the coiled-coil domain are not only involved in the interaction with phosphatidic acid, but also regulate the interaction of CIN85 with c-Cbl. CIN85 also interacts with ESCRT components for protein sorting in endosomes. These CIN85-protein and CIN85-lipid interactions enable CIN85 to link EGFR-Cbl endocytic complex with fusible membrane during EGFR endocytosis and subsequently to facilitate ESCRT formation on endosomal membrane for EGFR sorting and degradation.

Background: Trichomonas vaginalis, a flagellated protozoan, is the agent responsible for trichomoniasis, the most common nonviral sexually transmitted infection worldwide. A reported 200 million cases are documented each year with far more cases going unreported. However, T. vaginalis is disproportionality under studied, especially considering its basic metabolism. It has been reported that T. vaginalis does not grow on sucrose. Nevertheless, the T. vaginalis genome contains some 11 putative sucrose transporters and a putative β-fructofuranosidase (invertase). Thus, the machinery for both uptake and cleavage of sucrose appears to be present.

Results: We amplified the β-fructofuranosidase from T. vaginalis cDNA and cloned it into an Escherichia coli expression system. The expressed, purified protein was found to behave similarly to other known β-fructofuranosidases. The enzyme exhibited maximum activity at pH close to 5.0, with activity falling off rapidly at increased or decreased pH. It had a similar K(m) and V(max) to previously characterized enzymes using sucrose as a substrate, was also active towards raffinose, but had no detectable activity towards inulin.

Conclusions: T. vaginalis has the coding capacity to produce an active β-fructofuranosidase capable of hydrolyzing di- and trisaccharides containing a terminal, non-reducing fructose residue. Since we cloned this enzyme from cDNA, we know that the gene in question is transcribed. Furthermore, we could detect β-fructofuranosidase activity in T. vaginalis cell lysates. Therefore, the inability of the organism to utilize sucrose as a carbon source cannot be explained by an inability to degrade sucrose.

Background: Organic solute carrier partner 1 (OSCP1) is known to facilitate the transport of various organic solutes into cells and reported to play a role in cell growth and cell differentiation. Moreover, OSCP1 is known as a tumor suppressor gene that is frequently down-expressed in nasopharyngeal carcinomas and acute myeloid leukemia. However, the underlying mechanisms of action remain unclear and the subcellular localization of OSCP1 has yet to be determined in detail.

Results: Drosophila contains a single orthologue of OSCP1 (dOSCP1) that shares 58% homology with its human counterpart. To study the expression pattern and subcellular localization of dOSCP1, we prepared a specific antibody. Subcellular localization analyses of dOSCP1 with these revealed localization in the plasma membrane, endoplasmic reticulum, Golgi apparatus and mitochondria, but no detection in cytosol. dOSCP1 signals were also detected in the nucleus, although at weaker intensity than in plasma membranes and subcellular organelles. In addition, native polyacrylamide gel electrophoresis analysis with and without β-mercaptoethanol treatment revealed that recombinant dOSCP1 forms dimers and trimers in solution. The dimer form of dOSCP1 could also be detected by Western immunoblot analyses in third instar larval extracts.

Conclusions: The data revealed that dOSCP1 localizes not only in the plasma membrane but also in the nucleus, ER, Golgi apparatus and mitochondria. It is therefore conceivable that this protein may interact with various partners or form multimeric complexes with other proteins to play multiple roles in cells, providing clues to understanding the functions of dOSCP1 during Drosophila development.

Background: In order to understand the effects of FeS cluster attachment in [NiFe] hydrogenase, we undertook a study to substitute all 12 amino acid positions normally ligating the three FeS clusters in the hydrogenase small subunit. Using the hydrogenase from Alteromonas macleodii "deep ecotype" as a model, we substituted one of four amino acids (Asp, His, Asn, Gln) at each of the 12 ligating positions because these amino acids are alternative coordinating residues in otherwise conserved-cysteine positions found in a broad survey of NiFe hydrogenase sequences. We also hoped to discover an enzyme with elevated hydrogen evolution activity relative to a previously reported "G1" (H230C/P285C) improved enzyme in which the medial FeS cluster Pro and the distal FeS cluster His were each substituted for Cys.

Results: Among all the substitutions screened, aspartic acid substitutions were generally well-tolerated, and examination suggests that the observed deficiency in enzyme activity may be largely due to misprocessing of the small subunit of the enzyme. Alignment of hydrogenase sequences from sequence databases revealed many rare substitutions; the five substitutions present in databases that we tested all exhibited measurable hydrogen evolution activity. Select substitutions were purified and tested, supporting the results of the screening assay. Analysis of these results confirms the importance of small subunit processing. Normalizing activity to quantity of mature small subunit, indicative of total enzyme maturation, weakly suggests an improvement over the "G1" enzyme.

Conclusions: We have comprehensively screened 48 amino acid substitutions of the hydrogenase from A. macleodii "deep ecotype", to understand non-canonical ligations of amino acids to FeS clusters and to improve hydrogen evolution activity of this class of hydrogenase. Our studies show that non-canonical ligations can be functional and also suggests a new limiting factor in the production of active enzyme.

Background: Human malaria parasite infection and its control is a global challenge which is responsible for ~0.65 million deaths every year globally. The emergence of drug resistant malaria parasite is another challenge to fight with malaria. Enormous efforts are being made to identify suitable drug targets in order to develop newer classes of drug. Helicases play crucial roles in DNA metabolism and have been proposed as therapeutic targets for cancer therapy as well as viral and parasitic infections. Genome wide analysis revealed that Plasmodium falciparum possesses UvrD helicase, which is absent in the human host.

Results: Recently the biochemical characterization of P. falciparum UvrD helicase revealed that N-terminal UvrD (PfUDN) hydrolyses ATP, translocates in 3' to 5' direction and interacts with MLH to modulate each other's activity. In this follow up study, further characterization of P. falciparum UvrD helicase is presented. Here, we screened the effect of various DNA interacting compounds on the ATPase and helicase activity of PfUDN. This study resulted into the identification of daunorubicin (daunomycin), netropsin, nogalamycin, and ethidium bromide as the potential inhibitor molecules for the biochemical activities of PfUDN with IC50 values ranging from ~3.0 to ~5.0 μM. Interestingly etoposide did not inhibit the ATPase activity but considerable inhibition of unwinding activity was observed at 20 μM. Further study for analyzing the importance of PfUvrD enzyme in parasite growth revealed that PfUvrD is crucial/important for its growth ex-vivo.

Conclusions: As PfUvrD is absent in human hence on the basis of this study we propose PfUvrD as suitable drug target to control malaria. Some of the PfUvrD inhibitors identified in the present study can be utilized to further design novel and specific inhibitor molecules.

Background: The gene that encodes laforin, a dual-specificity phosphatase with a carbohydrate-binding module, is mutated in Lafora disease (LD). LD is an autosomal recessive, fatal progressive myoclonus epilepsy characterized by the intracellular buildup of insoluble, hyperphosphorylated glycogen-like particles, called Lafora bodies. Laforin dephosphorylates glycogen and other glucans in vitro, but the structural basis of its activity remains unknown. Recombinant human laforin when expressed in and purified from E. coli is largely insoluble and prone to aggregation and precipitation. Identification of a laforin ortholog that is more soluble and stable in vitro would circumvent this issue.

Results: In this study, we cloned multiple laforin orthologs, established a purification scheme for each, and tested their solubility and stability. Gallus gallus (Gg) laforin is more stable in vitro than human laforin, Gg-laforin is largely monomeric, and it possesses carbohydrate binding and phosphatase activity similar to human laforin.

Conclusions: Gg-laforin is more soluble and stable than human laforin in vitro, and possesses similar activity as a glucan phosphatase. Therefore, it can be used to model human laforin in structure-function studies. We have established a protocol for purifying recombinant Gg-laforin in sufficient quantity for crystallographic and other biophysical analyses, in order to better understand the function of laforin and define the molecular mechanisms of Lafora disease.