benzyloxycarbonylleucyl-leucyl-leucine-aldehyde and 25-hydroxycholesterol

ORP7 is a member of oxysterol-binding protein (OSBP) family, the function of which has remained obscure. In this study, we identified by yeast two-hybrid screening an interaction partner of ORP7, GATE-16, which (i) regulates Golgi SNARE of 28kDa (GS28) function and stability, and (ii) plays a role in autophagosome biogenesis. The interaction was confirmed by bimolecular fluorescence complementation (BiFC) assay in living cells. The interacting regions were delineated within aa 1-142 of ORP7 and aa 30-117 of GATE-16. ORP7 knock-down in 293A cells resulted in a 40% increase of GS28 protein while ORP7 overexpression had the opposite effect (25% decrease of GS28). We show evidence that the regulation of GS28 by ORP7 does not occur at the level of transcription, but involves degradation of GS28 on proteasomes. Truncated ORP7 that lacks the GATE-16 binding region failed to affect GS28 stability, evidencing for specificity of the observed effect. Similar to ORP7 overexpression, treatment of cells with 25-hydroxycholesterol (25-OH) resulted in GS28 destabilization, which was potentiated by excess ORP7 and inhibited by ORP7 silencing. Overexpression of ORP7 led in most cells to formation of vacuolar structures positive for RFP-LC3, thus representing autophagic elements. Also GATE-16 was found in the vacuolar ORP7-positive elements, suggesting that excess ORP7 increases entrapment of GATE-16 in autophagosomes. Taken together, our results suggest that ORP7 negatively regulates GS28 protein stability via sequestration of GATE-16, and may mediate the effect of 25-OH on GS28 and Golgi function.

Topics: Adaptor Proteins, Signal Transducing; Autophagy-Related Protein 8 Family; Binding Sites; Carrier Proteins; Cytoplasm; Gene Expression; Golgi Apparatus; HEK293 Cells; Humans; Hydroxycholesterols; Leupeptins; Microfilament Proteins; Phagosomes; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Binding; Protein Interaction Domains and Motifs; Protein Interaction Mapping; Proteolysis; Qb-SNARE Proteins; Receptors, Steroid; RNA, Small Interfering; Sequence Deletion; Transfection; Two-Hybrid System Techniques

Akt plays a role in protecting macrophages from apoptosis induced by some oxysterols. Previously we observed enhanced degradation of Akt in P388D1 moncocyte/macrophages following treatment with 25-hydroxycholesterol (25-OH) or 7-ketocholesterol (7-KC). In the present report we examine the role of the ubiquitin proteasomal pathway in this process. We show that treatment with 25-OH or 7-KC results in the accumulation of poly-ubiquitinated Akt, an effect that is enhanced by co-treatment with the proteasome inhibitor MG-132. Modification of Akt by the addition of a Gly-Ala repeat (GAr), a domain known to block ubiquitin-dependent targeting of proteins to the proteasome, resulted in a chimeric protein that is resistant to turn-over induced by 25-OH or 7-KC and provides protection from apoptosis induced by these oxysterols. These results uncover a new aspect of oxysterol regulation of Akt in macrophages; oxysterol-stimulated poly-ubiquitination of Akt and degradation by the proteasomal pathway.

Topics: Animals; Apoptosis; Base Sequence; Cell Line, Tumor; Hydroxycholesterols; Ketocholesterols; Leukemia P388; Leupeptins; Macrophages; Mice; Plasmids; Protease Inhibitors; Proteasome Endopeptidase Complex; Proto-Oncogene Proteins c-akt; Recombinant Fusion Proteins; Transfection; Ubiquitination

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.

Topics: Animals; beta-Galactosidase; Bridged Bicyclo Compounds, Heterocyclic; Cell Line; Cholesterol; Cricetinae; Cycloheximide; Cysteine Proteinase Inhibitors; Farnesyl-Diphosphate Farnesyltransferase; Humans; Hydroxycholesterols; Hydroxymethylglutaryl CoA Reductases; Leupeptins; Lovastatin; Oligopeptides; Peptide Hydrolases; Precipitin Tests; Proteasome Endopeptidase Complex; Recombinant Fusion Proteins; Tricarboxylic Acids; Ubiquitins