The E3 Ubiquitin Ligase MARCH6 Degrades Squalene Monooxygenase and Affects 3-Hydroxy-3-Methyl-Glutaryl Coenzyme A Reductase and the Cholesterol Synthesis Pathway

Abstract

bThe 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.T he mevalonate pathway leading to cholesterol synthesis is controlled transcriptionally and, for more rapid shutdown, posttranslationally (1). The third step in the pathway, catalyzed by 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGCR), is generally regarded as the rate-limiting step in cholesterol synthesis and has been intensively studied (2, 3). However, squalene monooxygenase (SM) is a neglected rate-limiting enzyme in cholesterol synthesis downstream of HMGCR. A flavin monooxygenase located in the endoplasmic reticulum (ER), SM catalyzes the conversion of squalene into monooxidosqualene (MOS), the step in the mevalonate pathway preceding cyclization to form the steroid backbone. It can also act on its product to yield dioxidosqualene (DOS), the precursor for the potent oxysterol regulator 24(S),25-epoxycholesterol, which fine-tunes acute cholesterol synthesis (4). SM resides after the isoprenoid branch of the mevalonate pathway, committing products to sterol synthesis. This may allow differential control of cholesterol synthesis from that of essential nonsterol products (5).We recently reported that SM's activity is controlled at the posttranslational level via accelerated cholesterol-dependent ubiquitination and proteasomal degradation (5). This regulation requires the first 100 amino acids of the protein-a region that is highly conserved in vertebrates but lacking in lower organisms, such as yeast (Saccharomyces cerevisiae). Moreover, this sequence is sufficient to confer cholesterol-dependent turnover when fused to green fluorescent protein (GFP) (5). We have established that the process of degradation is distinct from the sterol-regulated ubiquitination and proteasomal degradation of HMGCR, as it does not require...

A Conserved C-terminal Element in the Yeast Doa10 and Human MARCH6 Ubiquitin Ligases Required for Selective Substrate Degradation

“…7B). Differences in exogenous expression levels of the WT and RING-dead MARCH6 proteins were reported previously (31,50) and are known to be due to differences in MARCH6 degradation (42). Our data suggest that the CTE is required for MARCH6 self-regulation through proteasome-dependent degradation, and both residues Gly-885 and Asn-890 are important for this.…”

Cholesterol-mediated Degradation of 7-Dehydrocholesterol Reductase Switches the Balance from Cholesterol to Vitamin D Synthesis

“…7A). We previously identified MARCH6 as an E3 ligase involved in the proteasomal degradation of SM and HMGCR (28). However, in this case we excluded its role in targeting DHCR7 for degradation (Fig.…”

“…7A). Considering that the E3 ligase MARCH6 can target both SM and HMGCR for degradation, we employed two independent siRNAs to knock down MARCH6, as we have done previously (28). However, DHCR7 degradation was not rescued (Fig.…”

“…Additionally, oxysterols were tested at concentrations shown to degrade HMGCR (28). Side-chain oxysterols, such as 25-hydroxycholesterol, induce HMGCR degradation (29), whereas steroid-ring oxysterols can induce SM degradation (16).…”

Navigating the Shallows and Rapids of Cholesterol Synthesis Downstream of HMGCR

“…While HMCGR is post-translationally regulated by non-cholesterol sterols, including 24,25-dihydrolanosterol (12) and oxysterols (13), SM is directly regulated by cholesterol itself (10), which results in its degradation via the ubiquitin-proteasome system. MARCH6 has been identified as the E3 ligase responsible for this targeted degradation (14,15). Cholesterol-dependent degradation of SM can be reversed by unsaturated fatty acids, which stabilise SM by preventing its polyubiquitination by MARCH6 (16).…”