The Substrate Specificity And Hydrolysis Kinetics Of Deubiquitinating Enzyme UCH37

Partâ… Ubiquitin C-terminal hydrolases (UCHs) are one of five sub-families of de-ubiquitinating enzymes (DUBs) that hydrolyze the C-terminal peptide bond of ubiquitin derivatives. UCH37 (also known as UCH-L5) is the only UCH family protease that interacts with the 19S proteasome regulatory complex and disassembles Lys48-linked polyubiquintin from the distal end of the chain. UCH-L1 and UCH-L3 do not appear to have tetraubiquitin hydrolysis activity. It has been shown that UCH37 is responsible for the ubiquitin isopetidase activity in the PA700 (19S) proteasome regulatory complex. The previous studies also showed that the UCH37, UCH-domain, UCH37-Adrm1, and UCH37-Adrm1-S1 do not appear to have di-ubiquitin hydrolysis activity. In those studies, they also found that the de-ubiquitin activity clearly was originated from the UCH domain. Our experiment suggested that the UCH-domain (1-227) not only recognizes ubiquitin and processes small adducts by itself, but also has di-ubiquitin isopeptidase activity. We are also interested in the hydrolysis kinetics of different types of di-ubiquitins. To this goal, we purified a series of proteins and synthsized Lys48-and Lys63-linked di-ubiquitins:UbF45W-K48R-Ub-G75AG76P-His6, UbF45W-K63R-Ub-G75AG76P-His6. Linear Ub2:UbF45W-Ub-G75AG76P-His6 was also prepared by direct experssion in prokaryotic cells. We applied these three di-ubiquitins as the substrates of the UCH37N. After reaction, the His6 tagged proteins or peptides were removed with Ni-NTA beads, the enzymatic activities of UCH37N were quantitatively determined by measuring the relative fluorescent intensity of the released product UbF45W. Our results show that UCH37N discriminates these three di-ubiquitins, suggesting that it might be related to its functions in vivo. Part II1. The main aim of the study was to determine the role of cerium in the amelioration of magnesium-deficiency effects in spinach plants. Spinach plants were cultivated in Hoagland's solution. They were subjected to magnesium deficiency and to cerium chloride administered in the magnesium-present media and magnesium-deficient media. Spinach plants grown in the magnesium-present media and magnesium-deficient media were measured for key enzyme activities involved in nitrogen metabolism such as nitrate reductase, nitrite reductase, glutamate dehydrogenase, glutamate synthase, urease, glutamic-pyruvic transaminase, and glutamic-oxaloace protease transaminase. As the nitrogen metabolism in spinach was significantly inhibited by magnesium deficiency, it caused a significant reduction of spinach plant weight, leaf turning chlorosis. However, cerium treatment grown in magnesium-deficiency media significantly promoted the activities of the key enzymes as well as the contents of the free amino acids, chlorophyll, soluble protein, and spinach growth. It implied that Ce3+ could partly substitute for magnesium to facilitate the transformation from inorganic nitrogen to organic nitrogen, leading to the improvement of spinach growth2. Magnesium-deficiency conditions applied to spinach cultures caused an oxidative stress status in spinach chloroplast monitored by an increase in reactive oxygen species (ROS) accumulation. The enhancement of lipids peroxide of spinach chloroplast grown in magnesium-deficiency media suggested an oxidative attack that was activated by a reduction of antioxidative defense mechanism measured by analysing the activities of superoxide dismutase(SOD), catalase(CAT), ascorbate peroxidase(APX), guaiacol peroxidase(GPX) and glutathione reductase(GR), as well as antioxidants such as carotenoids and glutathione content. As the antioxidative response of chloroplast was reduced in spinach grown in magnesium-deficiency media, it caused a significant reduction of spinach plant weight, old leaves turning chlorosis. However, cerium treatment grown in magnesium-deficiency conditions decreased the malondialdehyde (MDA) and ROS, and increased activities of the antioxidative defense system, and improved spinach growth. Together, the experimental study implied that cerium could partly substitute for magnesium and increase the oxidative stress-resistance of spinach chloroplast grown in magnesium-deficiency conditions, but the mechanisms need further study.