A one-step specific assay for continuous detection of sirtuin 2 activity

Sirtuins (SIRTs) are nicotinamide adenine dinucleotide (NAD)-dependent histone deacetylases with diverse physiological functions. A variety of small molecules have been developed to interrogate the physiological function of SIRTs. Therefore, it is desirable to establish efficient and convenient assays to screen SIRTs modulators. In this study, we designed a series of fluorescent nonapeptide probes derived from substrates of SIRT1-SIRT3. Fluorescence increment of these probes is based on SIRT-mediated removal of the acyl side chain with fluorophore, which makes this system free of lysine-recognizing protease. Comparing the reaction of these fluorescent nonapeptide substrates with SIRT1-SIRT3 and SIRT6, it was confirmed that this assessment system was the most suitable for SIRT2 activity detection. Thus, SIRT2 was used to modify substrates by truncating the amino acids or lysine side chain of nonapeptide. Finally, two specific and efficient fluorescent probes for SIRT2, ne-D9 and ne-K4a, were developed. Evaluation of the results revealed that ne-K4a based assay was more suitable for modulators screening , while the other specific substrate ne-D9 was stable in cell lysate and could detect the activity of SIRT2 in the same. In summary, this study presents a novel strategy for detecting SIRT2 activity and in cell lysate.

Molecular and Biochemical Characterization of Opisthorchis viverrini Calreticulin

Calreticulin (CALR), a multifunctional protein thoroughly researched in mammals, comprises N-, P-, and C-domain and has roles in calcium homeostasis, chaperoning, clearance of apoptotic cells, cell adhesion, and also angiogenesis. In this study, the spatial and temporal expression patterns of the Opisthorchis viverrini CALR gene were analyzed, and calcium-binding and chaperoning properties of recombinant O. viverrini CALR (OvCALR) investigated. OvCALR mRNA was detected from the newly excysted juvenile to the mature parasite by RT-PCR while specific antibodies showed a wide distribution of the protein. OvCALR was localized in tegumental cell bodies, testes, ovary, eggs, Mehlis’ gland, prostate gland, and vitelline cells of the mature parasite. Recombinant OvCALR showed an in vitro suppressive effect on the thermal aggregation of citrate synthase. The recombinant OvCALR C-domain showed a mobility shift in native gel electrophoresis in the presence of calcium. The results imply that OvCALR has comparable function to the mammalian homolog as a calcium-binding molecular chaperone. Inferred from the observed strong immunostaining of the reproductive tissues, OvCALR should be important for reproduction and might be an interesting target to disrupt parasite fecundity. Transacetylase activity of OvCALR as reported for calreticulin of Haemonchus contortus could not be observed.