Ika Kristiana

Cholesterol-Dependent Degradation of Squalene Monooxygenase, a Control Point in Cholesterol Synthesis beyond HMG-CoA Reductase

Exquisite control of cholesterol synthesis is crucial for maintaining homeostasis of this vital yet potentially toxic lipid. Squalene monooxygenase (SM) catalyzes the first oxygenation step in cholesterol synthesis, acting on squalene before cyclization into the basic steroid structure. Using model cell systems, we found that cholesterol caused the accumulation of the substrate squalene, suggesting that SM may serve as a flux-controlling enzyme beyond 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR, considered as rate limiting). Cholesterol accelerated the proteasomal degradation of SM which required the N-terminal domain, partially conserved in vertebrates but not in lower organisms. Unlike HMGR, SM degradation is not mediated by Insig, 24,25-dihydrolanosterol, or side-chain oxysterols, but rather by cholesterol itself. Importantly, SMs N-terminal domain conferred cholesterol-regulated turnover on heterologous fusion proteins. Furthermore, proteasomal inhibition almost totally eliminated squalene accumulation, highlighting the importance of this degradation mechanism for the control of SM and suggesting this as a possible control point in cholesterol synthesis.

The E3 ubiquitin ligase MARCH6 degrades squalene monooxygenase and affects 3-hydroxy-3-methyl-glutaryl coenzyme a reductase and the cholesterol synthesis pathway

ABSTRACT The mevalonate pathway is used by cells to produce sterol and nonsterol metabolites and is subject to tight metabolic regulation. We recently reported that squalene monooxygenase (SM), an enzyme controlling a rate-limiting step in cholesterol biosynthesis, is subject to cholesterol-dependent proteasomal degradation. However, the E3-ubiquitin (E3) ligase mediating this effect was not established. Using a candidate approach, we identify the E3 ligase membrane-associated RING finger 6 (MARCH6, also known as TEB4) as the ligase controlling degradation of SM. We find that MARCH6 and SM physically interact, and consistent with MARCH6 acting as an E3 ligase, its overexpression reduces SM abundance in a RING-dependent manner. Reciprocally, knockdown of MARCH6 increases the level of SM protein and prevents its cholesterol-regulated degradation. Additionally, this increases cell-associated SM activity but is unexpectedly accompanied by increased flux upstream of SM. Prompted by this observation, we found that knockdown of MARCH6 also controls the level of 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGCR) in hepatocytes and model cell lines. In conclusion, MARCH6 controls abundance of both SM and HMGCR, establishing it as a major regulator of flux through the cholesterol synthesis pathway.

Cholesterol Homeostasis in Two Commonly Used Human Prostate Cancer Cell-Lines, LNCaP and PC-3

Background Recently, there has been renewed interest in the link between cholesterol and prostate cancer. It has been previously reported that in vitro, prostate cancer cells lack sterol-mediated feedback regulation of the major transcription factor in cholesterol homeostasis, sterol-regulatory element binding protein 2 (SREBP-2). This could explain the accumulation of cholesterol observed in clinical prostate cancers. Consequently, perturbed feedback regulation to increased sterol levels has become a pervasive concept in the prostate cancer setting. Here, we aimed to explore this in greater depth. Methodology/Principal Findings After altering the cellular cholesterol status in LNCaP and PC-3 prostate cancer cells, we examined SREBP-2 processing, downstream effects on promoter activity and expression of SREBP-2 target genes, and functional activity (low-density lipoprotein uptake, cholesterol synthesis). In doing so, we observed that LNCaP and PC-3 cells were sensitive to increased sterol levels. In contrast, lowering cholesterol levels via statin treatment generated a greater response in LNCaP cells than PC-3 cells. This highlighted an important difference between these cell-lines: basal SREBP-2 activity appeared to be higher in PC-3 cells, reducing sensitivity to decreased cholesterol levels. Conclusion/Significance Thus, prostate cancer cells are sensitive to changing sterol levels in vitro, but the extent of this regulation differs between prostate cancer cell-lines. These results shed new light on the regulation of cholesterol metabolism in two commonly used prostate cancer cell-lines, and emphasize the importance of establishing whether or not cholesterol homeostasis is perturbed in prostate cancer in vivo.

Antipsychotic drugs upregulate lipogenic gene expression by disrupting intracellular trafficking of lipoprotein-derived cholesterol.

Antipsychotic drugs (APDs) have been reported to induce lipogenic genes. This has been proposed to contribute to their efficacy in treating schizophrenia and other psychiatric disorders, as well as the metabolic side effects often associated with these drugs. The precise mechanism for the lipogenic effects of APDs is unknown, but is believed to involve increased activation of the lipogenic transcription factors, such as sterol regulatory element binding proteins (SREBPs). In a series of experiments in a model cell line, we found that a panel of typical and atypical APDs inhibited transport of lipoprotein-derived cholesterol to the endoplasmic reticulum (ER), which houses the cholesterol homeostatic machinery. APDs belong to the class of cationic amphiphiles and as has been shown for other amphiphiles, caused lipoprotein-derived cholesterol to accumulate intracellularly, preventing it from being esterified in the ER and suppressing SREBP activation. APDs did not activate the liver X receptor, another transcription factor involved in lipogenesis. However, these drugs markedly reduced cholesterol synthesis. This paradoxical result indicates that the upregulation of SREBP-target genes by APDs may not translate to increased cellular cholesterol levels. In conclusion, we have determined that APDs disrupt intracellular trafficking and synthesis of cholesterol, which may have important clinical ramifications.

The endogenous regulator 24(S),25-epoxycholesterol inhibits cholesterol synthesis at DHCR24 (Seladin-1)

The oxysterol 24(S),25-epoxycholesterol (24,25EC) can affect cholesterol metabolism at multiple points. Previously, we proposed that 24,25EC has an especially significant role in fine-tuning cholesterol synthesis, since it parallels cholesterol production, and without it, acute cholesterol synthesis is exaggerated. 24,25EC is structurally similar to desmosterol, a substrate for the enzyme 3β-hydroxysterol ∆(24)-reductase (DHCR24, also called Seladin-1) which catalyzes a final step in cholesterol synthesis. In this study, we reveal a novel mode by which 24,25EC can regulate cholesterol synthesis, by interfering with DHCR24, resulting in the rapid accumulation of the substrate desmosterol, at the expense of cholesterol. This effect was independent of DHCR24 protein levels, and was observed in multiple mammalian cell-lines, including those of hepatic and neuronal origin. Conversely, overexpression of DHCR24 blunted the inhibition by 24,25EC. We also determined that the specificity of this effect was restricted to certain side-chain oxysterols, notably those oxygenated at C-25. Importantly, endogenous levels of 24,25EC, manipulated by genetic and pharmacological methods, were sufficient to reduce DHCR24 activity. Together, our work introduces a novel role for 24,25EC in cholesterol homeostasis, through its rapid inhibition of cholesterol synthesis at DHCR24. Also, our work provides new insights into a little studied area, the post-transcriptional regulation of DHCR24, an important enzyme in human health and disease.

Different kinetics of cholesterol delivery to components of the cholesterol homeostatic machinery: Implications for cholesterol trafficking to the endoplasmic reticulum

Previously, using an oxysterol to induce cholesterol trafficking to the Endoplasmic Reticulum (ER), we reported a dissociation between cholesterol transport to two important cholesterol regulatory components in the ER: the cholesterol esterifying enzyme ACAT (Acyl CoA:Cholesterol Acyltransferase) and the membrane-bound transcription factor SREBP (Sterol Regulatory Element Binding Protein) (X. Du, Y.H. Pham and A.J. Brown, Effects of 25-hydroxycholesterol on cholesterol esterification and SREBP processing are dissociable: implications for cholesterol movement to the regulatory pool in the endoplasmic reticulum, J. Biol Chem. 279 (2004) 47010-47016). Here, we employed low-density lipoprotein (LDL) as a more physiologically-relevant mode of cholesterol delivery, and compared cholesterol transport to ACAT (determined by esterification) and SREBP (assessed by processing) in mutant Chinese Hamster Ovary cells that have cholesterol-trafficking defects (including Niemann-Pick type C). We showed clear differences in kinetics between the two, with impaired cholesterol trafficking to SREBP being resolved more rapidly than to ACAT. This is unlikely to be due to a reduced threshold of cholesterol sensed by the SREBP system relative to ACAT, since both responded to LDL-derived cholesterol within 2 h whereas the divergence observed between the two was prolonged (>20 h). Furthermore, ACAT inhibition did not expand the ER regulatory pool of cholesterol as judged by unaltered sensitivity of SREBP processing to LDL. Collectively, our data favor the contention that there are different cholesterol pools and/or transport pathways which feed ACAT and SREBP within the ER.

Signaling regulates activity of DHCR24, the final enzyme in cholesterol synthesis

The role of signaling in regulating cholesterol homeostasis is gradually becoming more widely recognized. Here, we explored how kinases and phosphorylation sites regulate the activity of the enzyme involved in the final step of cholesterol synthesis, 3β-hydroxysterol Δ24-reductase (DHCR24). Many factors are known to regulate DHCR24 transcriptionally, but little is known about its posttranslational regulation. We developed a system to specifically test human ectopic DHCR24 activity in a model cell-line (Chinese hamster ovary-7) using siRNA targeted only to hamster DHCR24, thus ensuring that all activity could be attributed to the human enzyme. We determined the effect of known phosphorylation sites and found that mutating certain residues (T110, Y299, and Y507) inhibited DHCR24 activity. In addition, inhibitors of protein kinase C ablated DHCR24 activity, although not through a known phosphorylation site. Our data indicate a novel mechanism whereby DHCR24 activity is regulated by signaling.

Cholesterol through the Looking Glass ABILITY OF ITS ENANTIOMER ALSO TO ELICIT HOMEOSTATIC RESPONSES

Background: Cholesterol is believed to be sensed via direct binding to the homeostatic machinery rather than by altering membrane properties. Results: Enantiomeric cholesterol, which exerts membrane effects but not specific interactions, also elicited homeostatic responses. Conclusion: Cholesterol mediates its own homeostasis through changing membrane properties in addition to specific cholesterol-protein binding. Significance: This work indicates a significant role of membrane effects in cholesterol homeostasis. How cholesterol is sensed to maintain homeostasis has been explained by direct binding to a specific protein, Scap, or through altering the physical properties of the membrane. The enantiomer of cholesterol (ent-cholesterol) is a valuable tool in distinguishing between these two models because it shares nonspecific membrane effects with native cholesterol (nat-cholesterol), but not specific binding interactions. This is the first study to compare ent- and nat-cholesterol directly on major molecular parameters of cholesterol homeostasis. We found that ent-cholesterol suppressed activation of the master transcriptional regulator of cholesterol metabolism, SREBP-2, almost as effectively as nat-cholesterol. Importantly, ent-cholesterol induced a conformational change in the cholesterol-sensing protein Scap in isolated membranes in vitro, even when steps were taken to eliminate potential confounding effects from endogenous cholesterol. Ent-cholesterol also accelerated proteasomal degradation of the key cholesterol biosynthetic enzyme, squalene monooxygenase. Together, these findings provide compelling evidence that cholesterol maintains its own homeostasis not only via direct protein interactions, but also by altering membrane properties.

Squalene mono-oxygenase, a key enzyme in cholesterol synthesis, is stabilized by unsaturated fatty acids.

SM (squalene mono-oxygenase) catalyses the first oxygenation step in cholesterol synthesis, immediately before the formation of the steroid backbone at lanosterol. SM is an important control point in the pathway, and is regulated at the post-translational level by accelerated cholesterol-dependent ubiquitination and proteasomal degradation, which is associated with the accumulation of squalene. Using model cell systems, we report that SM is stabilized by unsaturated fatty acids. Treatment with unsaturated fatty acids such as oleate, but not saturated fatty acids, increased protein levels of SM or SM-N100-GFP (the first 100 amino acids of SM fused to GFP) at the post-translational level and partially overcame cholesterol-dependent degradation, as well as reversing cholesterol-dependent squalene accumulation. Maximum stabilization required activation of fatty acids, but not triacylglycerol or phosphatidylcholine synthesis. The mechanism of oleate-mediated stabilization appeared to occur through reduced ubiquitination by the E3 ubiquitin ligase MARCH6. Stabilization of a cholesterol biosynthetic enzyme by unsaturated fatty acids may help maintain a constant cholesterol/phospholipid ratio.