Membrane association of glutathione S-transferase mGSTA4-4, an enzyme that metabolizes lipid peroxidation products.

Lipid peroxidation products have signaling functions and at higher concentrations are toxic and may trigger cell death. The compounds are metabolized predominantly by glutathione S-transferases exemplified by mGSTA4-4, an enzyme highly efficient in glutathione conjugation of 4-hydroxyalkenals, and possessing glutathione peroxidase activity toward phospholipid hydroperoxides. mGSTA4-4 belongs to the predominant group of "canonical" glutathione S-transferases that are soluble and generally localized in the cytoplasm. The intracellular localization of mGSTA4-4 was examined in hepatocytes of normal mouse liver and in transfected HepG2 cells by fluorescence microscopy and digital deconvolution. mGSTA4-4 was found to be predominantly localized at or near the plasma membrane in transfected HepG2 cells, as well as in hepatocytes endogenously expressing the protein. In vitro, mGSTA4-4 associated with liposomes, and this interaction was potentiated when the liposomes contained negatively charged phospholipids. Mutating lysine 115 to glutamic acid resulted in a loss of the plasma membrane targeting of mGSTA4-4 as well as in a significant reduction of its binding to liposomes in vitro. These data suggest preferential targeting of mGSTA4-4 to the plasma membrane that may contain the major substrate(s) for this enzyme. Lysine 115 is critically important for the membrane association of mGSTA4-4, most likely by entering into an electrostatic interaction with negatively charged phospholipid headgroups.     

A new method of preparing Ca2+-ATPase from sarcoplasmic reticulum: extraction with octylglucoside.

A fast method for preparing Ca2+-ATPase from rabbit muscle sarcoplasmic reticulum was devised. The method involves extracting extrinsic membrane proteins with the non-ionic detergent octylglucoside at high salt concentration. A Ca2+-ATPase of consistently high specific activity (about 25 mumoles/mg.min) is found in the insoluble residue. The method was optimized with respect to the concentrations of detergent and salt, pH, and other extraction conditions. By the criteria of the protein pattern in SDS-polyacrylamide gel electrophoresis, dependence of the hydrolytic activity on the presence of Ca2+, and the phosphoprotein formation, the preparation is identical with the Ca2+-ATPase isolated previously by MacLennan [10] and other authors. The main advantages of the new method are its rapidity, its reliability, and the high specific activity of the purified enzyme.     

Transport of glutathione-conjugates in human erythrocytes

The last step of detoxification of both endogenous and environmental toxicants is typically a conjugation that produces a bulky hydrophilic molecule. The excretion of such conjugates out of cells is of sufficient biological importance to have led to the evolution of ATP-driven export pumps for this purpose. The substrate specificity of such transporters is broad, and in some cases it has been shown to include not only anionic conjugates but also neutral or weakly cationic drugs. In the present article, we review the molecular identity, functional and structural characteristics of these pumps, mainly on the example of human erythrocytes, and discuss their physiological role in detoxification and in the multidrug resistance phenotype of cancer cells.     

CENP-C is a blueprint for constitutive centromere–associated network assembly within human kinetochores

Kinetochores are multisubunit complexes that assemble on centromeres to bind spindle microtubules and promote faithful chromosome segregation during cell division. A 16-subunit complex named the constitutive centromere–associated network (CCAN) creates the centromere–kinetochore interface. CENP-C, a CCAN subunit, is crucial for kinetochore assembly because it links centromeres with the microtubule-binding interface of kinetochores. The role of CENP-C in CCAN organization, on the other hand, had been incompletely understood. In this paper, we combined biochemical reconstitution and cellular investigations to unveil how CENP-C promotes kinetochore targeting of other CCAN subunits. The so-called PEST domain in the N-terminal half of CENP-C interacted directly with the four-subunit CCAN subcomplex CENP-HIKM. We identified crucial determinants of this interaction whose mutation prevented kinetochore localization of CENP-HIKM and of CENP-TW, another CCAN subcomplex. When considered together with previous observations, our data point to CENP-C as a blueprint for kinetochore assembly.     

Lifespan extension in hypomorphic daf-2 mutants of Caenorhabditis elegans is partially mediated by glutathione transferase CeGSTP2-2

Electrophilic stress caused by lipid peroxidation products such as 4-hydroxynonenal (4-HNE) and/or related compounds may contribute to aging. The major mode of 4-HNE metabolism involves glutathione conjugation catalyzed by specialized glutathione transferases. We have previously shown that glutathione transferase CeGSTP2-2, the product of the Caenorhabditis elegans gst-10 gene, has the ability to conjugate 4-HNE, and that its overexpression extends lifespan of C. elegans. We now demonstrate that the expression level of CeGSTP2-2 correlates highly with lifespan in a series of hypomorphic daf-2 mutants of C. elegans. The overexpression of CeGSTP2-2 in daf-2 is abrogated in daf-16; daf-2 mutants, indicating that expression of the gst-10 gene is modulated by insulin-like growth factor signaling. To determine whether the relationship between CeGSTP2-2 and lifespan is causal, we used RNAi to knock down CeGSTP2-2. Treatment with gst-10-specific dsRNA decreased CeGSTP2-2 protein in wild-type N2 and in daf-2 strains to an approximately equal level. The ability to conjugate 4-HNE was similarly decreased by RNAi, suggesting that the increment of that activity in daf-2 over N2 is due largely to the overexpression of CeGSTP2-2. RNAi-mediated knock-down of CeGSTP2-2 led to an increased susceptibility to 4-HNE, paraquat, and heat shock, and to a shortening of lifespan by 13% in both N2 and daf-2 strains. These results indicate that CeGSTP2-2 significantly contributes to the maintenance of the soma, and that this function is augmented in daf-2 mutants concordantly with other longevity assurance genes, probably via insulin-like growth factor signaling.     

Structural basis for catalytic differences between alpha class human glutathione transferases hGSTA1-1 and hGSTA2-2 for glutathione conjugation of environmental carcinogen benzo[a]pyrene-7,8-diol-9,10-epoxide

The ultimate diol epoxide carcinogens derived from polycyclic aromatic hydrocarbons, such as benzo[a]pyrene (BP), are metabolized primarily by glutathione (GSH) conjugation reaction catalyzed by GSH transferases (GSTs). In human liver and probably lung, the alpha class GSTs are likely to be responsible for the majority of this reaction because of their high abundance. The catalytic efficiency for GSH conjugation of the carcinogenic (+)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide [(+)-anti-BPDE] is more than 5-fold higher for hGSTA1-1 than for hGSTA2-2. Here, we demonstrate that mutation of isoleucine-11 of hGSTA2-2, a residue located in the hydrophobic substrate-binding site (H-site) of the enzyme, to alanine (which is present in the same position in hGSTA1-1) results in about a 7-fold increase in catalytic efficiency for (+)-anti-BPDE-GSH conjugation. Thus, a single amino acid substitution is sufficient to convert hGSTA2-2 to a protein that matches hGSTA1-1 in its catalytic efficiency. The increased catalytic efficiency of hGSTA2/I11A is accompanied by greater enantioselectivity for the carcinogenic (+)-anti-BPDE over (-)-anti-BPDE. Further remodeling of the H-site of hGSTA2-2 to resemble that of hGSTA1-1 (S9F, I11A, F110V, and S215A mutations, SIFS mutant) results in an enzyme whose catalytic efficiency is approximately 13.5-fold higher than that of the wild-type hGSTA2-2, and about 2.5-fold higher than that of the wild-type hGSTA1-1. The increased activity upon mutations can be rationalized by the interactions of the amino acid side chains with the substrate and the orientation of the substrate in the active site, as visualized by molecular modeling. Interestingly, the catalytic efficiency of hGSTA2-2 toward (-)-anti-BPDE was increased to a level close to that of hGSTA1-1 upon F110V, not I11A, mutation. Similar to (+)-anti-BPDE, however, the SIFS mutant was the most efficient enzyme for GSH conjugation of (-)-anti-BPDE.     

Biosynthesis of hydroxyl-linked glucuronides of short-chain bile acids by rat liver 3-hydroxysteroid UDP-glucuronosyltransferase

Microsomal preparations from livers of Sprague-Dawley rats catalyze the glucuronidation of 3 alpha-hydroxy-5 beta-H (3 alpha, 5 beta) short-chain bile acids (C20-C23), predominantly at the hydroxyl group, while the glucuronidation of 3 beta, 5 beta short-chain bile acids occurs exclusively at the carboxyl group. A similar pattern of conjugation was also observed in Wistar rats having normal levels of 3-hydroxysteroid UDP-glucuronosyltransferase. Significant reductions of formation rates for hydroxyl-linked, but not carboxyl-linked, short-chain bile acid glucuronides were observed in hepatic microsomes from Wistar rats with low 3-hydroxysteroid UDP-glucuronosyltransferase activity. 3-Hydroxysteroid UDP-glucuronosyltransferase, purified to homogeneity from Sprague-Dawley liver microsomes, catalyzed the 3-O-glucuronidation of 3 alpha, 5 beta C20-23 bile acids, as well as of lithocholic and isolithocholic acids (C24). The apparent Michaelis constants (KM) for short-chain bile acids were similar to the value obtained for androsterone. 3 alpha, 5 beta-C20 and 3 beta, 5 beta-C20 competitively inhibited glucuronidation of androsterone by the purified 3-hydroxysteroid UDP-glucuronosyltransferase. Purified 17 beta-hydroxysteroid and p-nitrophenol UDP-glucuronosyltransferases did not catalyze the glucuronidation of bile acids. In addition, none of the purified transferases catalyzed the formation of carboxyl-linked bile acid glucuronides. The results show that 3-hydroxysteroid UDP-glucuronosyltransferase, an enzyme specific for 3-hydroxyl groups of androgenic steroids and some conventional bile acids, also catalyzes the glucuronidation of 3 alpha-hydroxyl (but not carboxyl) groups of 3 alpha, 5 beta short-chain bile acids.     

Metabolism of lithocholic acid in the rat: formation of lithocholic acid 3-O-glucuronide in vivo

Milligram amounts of [3 beta-3H]lithocholic (3 alpha-hydroxy-5 beta-cholanoic) acid were administered by intravenous infusion to rats prepared with a biliary fistula. Analysis of sequential bile samples by thin-layer chromatography (TLC) demonstrated that lithocholic acid glucuronide was present in bile throughout the course of the experiments and that its secretion rate paralleled that of total isotope secretion. Initial confirmation of the identity of this metabolite was obtained by the recovery of labeled lithocholic acid after beta-glucuronidase hydrolysis of bile samples. For detailed analysis of biliary metabolites of [3H]lithocholic acid, pooled bile samples from infused rats were subjected to reversed-phase chromatography and four major labeled peaks were isolated. After complete deconjugation, the two major compounds in the combined first two peaks were identified as murideoxycholic (3 alpha, 6 beta-dihydroxy-5 beta-cholanoic) and beta-muricholic (3 alpha, 6 beta, 7 beta-trihydroxy-5 beta-cholanoic) acids and the third peak was identified as taurolithocholic acid. The major component of the fourth peak, after isolation, derivatization (to the methyl ester acetate), and purification by high pressure liquid chromatography (HPLC), was positively identified by proton nuclear magnetic resonance as lithocholic acid 3 alpha-O-(beta-D-glucuronide). These studies have shown, for the first time, that lithocholic acid glucuronide is a product of in vivo hepatic metabolism of lithocholic acid in the rat.     

C3 Rho-Inhibitor for Targeted Pharmacological Manipulation of Osteoclast-Like Cells

The C3 toxins from Clostridium botulinum (C3bot) and Clostridium limosum (C3lim) as well as C3-derived fusion proteins are selectively taken up into the cytosol of monocytes/macrophages where the C3-catalyzed ADP-ribosylation of Rho results in inhibition of Rho-signalling and characteristic morphological changes. Since the fusion toxin C2IN-C3lim was efficiently taken up into and inhibited proliferation of murine macrophage-like RAW 264.7 cells, its effects on RAW 264.7-derived osteoclasts were investigated. C2IN-C3lim was taken up into differentiated osteoclasts and decreased their resorption activity. In undifferentiated RAW 264.7 cells, C2IN-C3lim-treatment significantly decreased their differentiation into osteoclasts as determined by counting the multi-nucleated, TRAP-positive cells. This inhibitory effect was concentration- and time-dependent and most efficient when C2IN-C3lim was applied in the early stage of osteoclast-formation. A single-dose application of C2IN-C3lim at day 0 and its subsequent removal at day 1 reduced the number of osteoclasts in a comparable manner while C2IN-C3lim-application at later time points did not reduce the number of osteoclasts to a comparable degree. Control experiments with an enzymatically inactive C3 protein revealed that the ADP-ribosylation of Rho was essential for the observed effects. In conclusion, the results indicate that Rho-activity is crucial during the early phase of osteoclast-differentiation. Other bone cell types such as pre-osteoblastic cells were not affected by C2IN-C3lim. Due to their cell-type selective and specific mode of action, C3 proteins and C3-fusions might be valuable tools for targeted pharmacological manipulation of osteoclast formation and activity, which could lead to development of novel therapeutic strategies against osteoclast-associated diseases.