SPR Analysis of SUMO-Murine Rap1-Interacting Factor 1 C-Terminal Domain Interaction with G4.

One of the advantages of surface plasmon resonance is its sensitivity and real-time analyses performed by this method. These characteristics allow us to further investigate the interactions of challenging proteins like Rap1-interacting factor 1 (Rif1). Rif1 is a crucial protein responsible for regulating different cellular processes including DNA replication, repair, and transcription. Mammalian Rif1 is yet to be fully characterized, partly because it is predicted to be intrinsically disordered for a large portion of its polypeptide. This protein has recently been the target of research as a potential biomarker in many cancers. Therefore, finding its most potent interacting partner is of utmost importance. Previous studies showed Rif1's affinity towards structured DNAs and amongst them, T6G24 was superior. Recent studies have shown mouse Rif1 (muRif1) C-terminal domain's (CTD) role in binding to G-quadruplexes (G4). There were many concerns in investigating the Rif1 and G4 interaction, which can be minimized using SPR. Therefore, for the first time, we have assessed its binding with G4 at nano-molar concentrations with SPR which seems to be crucial for its binding analyses. Our results indicate that muRif1-CTD has a high affinity for this G4 sequence as it shows a very low KD (6 ± 1 nM).

Characterization of Interaction of the MBP-Tagged MuRif1-C-Terminal Domain with G-Quadruplex DNA by SPR.

One of the main players in the cell-specific replication timing pattern is Rap1 interacting factor-1 (Rif1). Rif1 protein consists of N-terminal and C-terminal domains and an intrinsically disordered region in between. It has been suggested that both N- and C-termini of Rif1 are capable of binding to DNA with particularly high affinity to cruciform DNA structures. In the present study, we expressed, solubilized, and purified the maltose-binding protein-tagged murine Rif1 C-terminal domain (MBP-muRif1-CTD). Biological activity of the purified protein was assessed by the electrophoretic mobility shift assay (EMSA) and surface plasmon resonance (SPR). Our results show that the MBP-muRif1-CTD binds G-quadruplex (G4) structure with high affinity (KD 19.0 ± 0.8 nM), as was previously suggested. This study is the first step in investigation of the interaction of MBP-Profinity eXact-muRif1-CTD and G4 by SPR.

G-quadruplex binding protein Rif1, a key regulator of replication timing.

DNA replication is spatially and temporally regulated during S phase to execute efficient and coordinated duplication of entire genome. Various epigenomic mechanisms operate to regulate the timing and locations of replication. Among them, Rif1 plays a major role to shape the 'replication domains' that dictate which segments of the genome are replicated when and where in the nuclei. Rif1 achieves this task by generating higher-order chromatin architecture near nuclear membrane and by recruiting a protein phosphatase. Rif1 is a G4 binding protein, and G4 binding activity of Rif1 is essential for replication timing regulation in fission yeast. In this article, we first summarize strategies by which cells regulate their replication timing and then describe how Rif1 and its interaction with G4 contribute to regulation of chromatin architecture and replication timing.

Enzyme(s) responsible for tellurite reducing activity in a moderately halophilic bacterium, Salinicoccus iranensis strain QW6.

Oxyanions of tellurium, like tellurate (TeO4 (2-)) and tellurite (TeO3 (2-)), are highly toxic for most microorganisms. There are a few reports on the bacterial tellurite resistance mechanism(s). Salinicoccus iranensis, a Gram-positive halophilic bacterium, shows high tellurite resistance and NADH-dependent tellurite reduction activity in vitro. Since little is known regarding TeO3 (2-) resistance mechanisms in halophilic microorganisms, here one of the enzymatic reduction activities presented in this microorganism is investigated. To enhance the enzymatic activity during purification, the effect of different parameters including time, inoculation, different pHs, different tellurite concentrations and different salts were optimized. We also examined the tellurite removal rates by diethyldithiocarbamate (DDTC) during optimization. In the culture medium the optimum conditions obtained showed that at 30 h, 2 % inoculum, pH 7.5, without tellurite and with 5 % NaCl (w/v) the highest enzyme activity and tellurite removal were observed. Results of the purification procedure done by hydroxyapatite batch-mode, ammonium sulfate precipitation, followed by phenyl-Sepharose and Sephadex G-100 column chromatography, showed that the enzyme consisted of three subunits with molecular masses of 135, 63 and 57 kDa. In addition to tellurite reduction activity, the enzyme was able to reduce nitrate too. Our study extends the knowledge regarding this process in halophilic microorganisms. Besides, this approach may suggest an application for the organism or the enzyme itself to be used for bioremediation of polluted areas with different contaminants due to its nitrate reductase activity.

Interaction of methocarbamol and yeast sucrase induces enzyme inhibition.

BACKGROUND: Methocarbamol is a skeletal muscle relaxant and is widely used to relieve pain in muscles. Many drugs may have interactions with each other when used at the same time. Yeast sucrase is taken as a drug by patients with congenital sucrase-isomaltase deficiency (CSID). METHODS: In this study, the interaction between methocarbamol and yeast sucrase was investigated. RESULTS: Our results showed that methocarbamol can inhibit sucrase activity and reduce the maximum reaction velocity (Vmax) of the enzyme by a non-competitive pattern. Measurement of IC50 and Ki of the drug revealed that methocarbamol did not bind the enzyme with high affinity. Fluorescence measurement showed that the drug binds to free enzyme and enzyme-substrate complexes that were accompanied by structural changes on the enzyme. Guaifenesin, which has a similar structure to methocarbamol, does not affect the activity of sucrase. CONCLUSIONS: Methocarbamol inhibits sucrase activity and its carbamate group plays a main role in the binding of drug to sucrase.

Ranitidine induces inhibition and structural changes in sucrase.

Ranitidine is an antagonist of histamine-2 (H(2)) receptor. It is employed to treat peptic ulcer and other conditions in which gastric acidity must be reduced. Sucrase is a hydrolytic enzyme that catalyzes the breakdown of sucrose to its monomer content. A liquid of yeast sucrase was developed for treatment of congenital sucrase-isomaltase deficiency (CSID) in human. In this study, the effect of ranitidine on yeast sucrase activity was investigated. Our results showed that ranitidine binds to sucrase and inhibits the enzyme in a noncompetitive manner. The K(i) and IC(50) values were measured to be about 2.3 and 2.2 mM, respectively. Fluorescence measurement showed conformational changes after binding of ranitidine to the enzyme. The fluorescence spectra showed that ranitidine could bind to both free enzyme and enzyme-substrate complex, which was accompanied with reduction of emission intensity and red shift production.

Inhibition of renal alkaline phosphatase by cimetidine.

Alkaline phosphatase (ALP) belongs to hydrolase group of enzymes. It is responsible for removing phosphate groups from many types of molecules, including nucleotides and proteins. Cimetidine (trade name Tagamet) is an antagonist of histamine H2-receptor that inhibits the production of gastric acid. Cimetidine is used for the treatment of gastrointestinal diseases. In this study the inhibitory effect of cimetidine on mouse renal ALP activity was investigated. Our results showed that cimetidine can inhibit ALP by uncompetitive inhibition. In the absence of inhibitor the V(max) and K(m) of the enzyme were found to be 13.7 mmol/mg prot.min and 0.25 mM, respectively. Both the Vmax and Km of the enzyme decreased with increasing cimetidine concentrations (0- 1.2 mM). The Ki and IC(50) of cimetidine were determined to be about 0.5 mM and 0.52 mM, respectively.