Molecular genetics of the ubiquitin-proteasome system: lessons from yeast.

Our studies with the yeast Saccharomyces cerevisiae have uncovered a number of general principles governing substrate selectivity and proteolysis by the ubiquitin-proteasome system. The initial work focused on the degradation of a transcription factor, the MATalpha2 repressor, but the pathways uncovered have a much broader range of targets. At least two distinct ubiquitination mechanisms contribute to alpha2 turnover. One of them depends on a large integral membrane ubiquitin ligase (E3) and a pair of ubiquitin-conjugating enzymes (E2s). The transmembrane E3 and E2 proteins must travel from their site of synthesis in the ER to the inner nuclear membrane in order to reach nuclear substrates such as alpha2. The 26S proteasome is responsible for alpha2 degradation, and several important features of proteasome assembly and active site formation were uncovered. Most recently, we have delineated major steps in 20S proteasome assembly and have also identified several novel 20S proteasome assembly factors. Surprisingly, alterations in 20S proteasome assembly lead to defects in the assembly of the proteasome regulatory particle (RP). The RP associates with the 20S proteasome to form the 26S proteasome. Our data suggest that the 20S proteasome can function as an assembly factor for the RP, which would make it the first such factor for RP assembly identified to date.

Ceramide-mediated apoptosis in lung epithelial cells is regulated by glutathione.

Reactive oxygen species (ROS) are mediators of lung injury, and glutathione (GSH) is the major nonprotein antioxidant that protects the cell from oxidative stress. We have recently shown that H(2)O(2) induces ceramide-mediated apoptosis in human lung epithelial cells. We hypothesized that ROS-mediated depletion of GSH plays a regulatory role in ceramide generation, and thus in the induction of apoptosis. Our present studies demonstrate that GSH at physiologic concentrations (1 to 10 mM) inhibits ceramide production in a time- and dose-dependent manner in A549 human alveolar epithelial cells. On the other hand, buthionine-sulfoximine-mediated depletion of intracellular GSH induces elevation of ceramide levels and apoptosis. In addition, GSH blocks H(2)O(2)-mediated induction of intracellular ceramide generation and apoptosis. These effects were not mimicked by oxidized GSH (GSSG) or other thiol antioxidants, such as dithiothreitol and 2-mercaptoethanol. Moreover, increase of intracellular H(2)O(2), mediated by inhibition of catalase by aminotriazole, also induces ceramide generation and apoptosis. These effects were blocked by N-acetylcysteine. Our results suggest that GSH depletion may be the link between oxidative stress and ceramide-mediated apoptosis in the lung.

The ubiquitin-proteasome pathway mediates the regulated degradation of mammalian 3-hydroxy-3-methylglutaryl-coenzyme A reductase.

3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR), the key regulatory enzyme in the mevalonate (MVA) pathway, is rapidly degraded in mammalian cells supplemented with sterols or MVA. This accelerated turnover was blocked by N-acetyl-leucyl-leucyl-norleucinal (ALLN), MG-132, and lactacystin, and to a lesser extent by N-acetyl-leucyl-leucyl-methional (ALLM), indicating the involvement of the 26 S proteasome. Proteasome inhibition led to enhanced accumulation of high molecular weight polyubiquitin conjugates of HMGR and of HMGal, a chimera between the membrane domain of HMGR and beta-galactosidase. Importantly, increased amounts of polyubiquitinated HMGR and HMGal were observed upon treating cells with sterols or MVA. Cycloheximide inhibited the sterol-stimulated degradation of HMGR concomitantly with a marked reduction in polyubiquitination of the enzyme. Inhibition of squalene synthase with zaragozic acid blocked the MVA- but not sterol-stimulated ubiquitination and degradation of HMGR. Thus, similar to yeast, the ubiquitin-proteasome pathway is involved in the metabolically regulated turnover of mammalian HMGR. Yet, the data indicate divergence between yeast and mammals and suggest distinct roles for sterol and nonsterol metabolic signals in the regulated ubiquitination and degradation of mammalian HMGR.

Impaired regulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase degradation in lovastatin-resistant cells.

L-90 cells were selected to grow in the presence of serum lipoproteins and 90 microM lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR). L-90 cells massively accumulate HMGR, a result of >10-fold amplification of the gene and 40-fold rise in mRNA, and also overexpress other enzymes of the mevalonate pathway. Western blot and promoter-luciferase analyses indicate that transcriptional regulation of sterol-responsive genes by 25-hydroxycholesterol or mevalonate is normal. Yet, none of these genes is regulated by lipoproteins, a result of severe impairment in the low density lipoprotein receptor pathway. Moreover, L-90 cells do not accelerate the degradation of HMGR or transfected HMGal chimera in response to 25-hydroxycholesterol or mevalonate. This aberrant phenotype persists when cells are grown without lovastatin for up to 37 days. The inability to regulate HMGR degradation is not due to its overproduction since in LP-90 cells, which were selected for lovastatin resistance in lipoprotein-deficient serum, HMGR is overexpressed, yet its turnover is regulated normally. Also, the rapid degradation of transfected alpha subunit of T cell receptor is markedly retarded in L-90 cells. These results show that in addition to gene amplification and overexpression of cholesterogenic enzymes, statin resistance can follow loss of regulated HMGR degradation.

Lipid accumulation in the ovaries of a marine shrimp penaeus semisulcatus (de haan)

By the end of oocyte development, the ovaries of Penaeus semisulcatus have accumulated almost equal amounts (approximately 16 mg lipid g-1 protein) of phospholipids and triacylglycerols. The phospholipids consist mainly of phosphatidylcholine (75-80 %) and phosphatidylethanolamine (20-25 %). Approximately 30 % of the total fatty acid content of both phospholipids and triacylglycerols is made up of polyunsaturated fatty acids. In fractions obtained by centrifugation of ovarian homogenates, most of the increase in levels of ovarian lipids during ovarian maturation was associated with an increase in triacylglycerol levels in the floating fat fraction and of phospholipids in the infranatant fraction. The presence of polyunsaturated fatty acids in the ovaries indicates the occurrence of lipid transport to the ovary during oocyte maturation. The gradual decrease in the relative abundance of polyunsaturated fatty acids as the ovaries matured supports previously published results suggesting intra-ovarian synthesis of saturated and mono-unsaturated fatty acids. Most of the lipids found in the female haemolymph (64.8 %) were recovered in the high-density lipoprotein fraction after density ultracentrifugation. The haemocyanin fraction recovered from this stage of fractionation contained substantial amounts of lipid (16.8 %) that could be removed by further sequential centrifugation at a higher NaBr density, leaving less than 0.9 % of the total haemolymph lipids associated with this fraction. While 16.2 % of the lipids were recovered from the very high-density lipoprotein fractions, these lipoproteins carried only 64-89 microg lipid mg-1 protein compared with 538.9 microg lipid mg-1 protein in the high-density lipoprotein fraction, indicating that the high-density lipoproteins are more likely to be the main transporters of lipids to the ovary. However, the contribution of very high-density lipoproteins to lipid transport cannot be ruled out at this stage. In this study, we present two models for lipid transport to the ovary based on the abundance of phospholipids and triacylglycerols in the haemolymph and on the amounts of polyunsaturated fatty acids accumulated within the ovary during vitellogenesis.

Isolation and characterization of the high-density lipoproteins from the hemolymph and ovary of the penaeid shrimp Penaeus semisulcatus (de Haan): apoproteins and lipids.

The high-density lipoproteins (HDLs) found in the male and female hemolymph of Penaeus semisulcatus de Haan were isolated by NaBr (1.22 g/ml) followed by sucrose gradient (5-25%) ultracentrifugation. The male HDL contained one protein, lipoprotein 1 (LP1), composed of one 110-kDa peptide subunit. The female HDL contained two proteins: 1) the LP1 that was immunoidentical to the male LP1 and was similarly composed of one 110-kDa peptide subunit and 2) vitellogenin (Vg), reacting positively with the rabbit antiserum generated against vitellin (Vt) that was isolated from vitellogenic ovaries. Both Vg and Vt consisted mainly of three polypeptide subunits (200, 120, and 80 kDa) as revealed by denatured PAGE and Western blot. The LP1 from males or females did not react with the Vt rabbit antiserum. Similarly, Vg and Vt did not react with the rabbit antiserum prepared against LP1. Phospholipids (PL) constituted 71-76% of the total lipids in the hemolymph and HDLs of both male and female hemolymph. Cholesterol (Ch) amounted to 17-20%, and small amounts (5%) of diacylglycerols (DAG) were also carried by these HDLs. Both the PL and DAG contained highly unsaturated fatty acids (20:5 omega 3 and 22:6 omega 3) that are transported from the food or hepatopancreas to the tissues, including the vitellogenic ovaries in females. In the present study we show for the first time the separate lipid composition of female LP1 and Vg and compare them with the lipids attached to the Vt. Vg had a lower lipid content than LP1 (540 and 1089 mg/g protein, respectively). Differences were also found in the relative abundance of PL, Ch, and DAG classes in the LP1 in comparison with Vg. Furthermore, small amounts (approximately 3.8%) of triacylglycerols (TAG) were found only in the hemolymph of vitellogenic females, and they were associated with the Vg. Although Vg and Vt were composed of similar polypeptides, their lipid composition was different Vt, in contrast to Vg, carried considerable amounts of TAG (approximately 22%) and only trace amounts of DAG. The significance of the TAG in the hemolymph of vitellogenic females is not known, and the functional relationship between Vg and Vt requires future extensive studies. Lipids were not detected in hemocyanin that was purified from clotted hemolymph.