INSIG, SCAP and HMG-CoA Reductase
Sterols and oxysterols regulate cholesterol synthesis by binding to SCAP or INSIG, and via SREBP-2 modulate the expression of the enzymes of the cholesterol biosynthesis pathway (Brown, Radhakrishnan & Goldstein, 2018). At times of cholesterol excess, cholesterol in the endoplasmic reticulum binds to SCAP, which is already in complex with SREBPs, and causes a conformational change in SCAP which results in binding of the SCAP-SREBP complex to the endoplasmic reticulum - anchor protein INSIG. The consequence of this event is retention of SREBP-2 in the endoplasmic reticulum and prevention of its transport by SCAP to the Golgi for processing to its active form as a transcription factor for the enzymes of the cholesterol biosynthesis pathway and for the LDLR (Goldstein, DeBose-Boyd & Brown, 2006; Sun, Seemann, Goldstein & Brown, 2007). Oxysterols also have a role in regulating cholesterol biosynthesis (Kandutsch, Chen & Heiniger, 1978), but in most cases by providing “fine tuning” of this regulation (Gill, Chow & Brown, 2008). The brain is an organ with high oxysterol concentrations (24S-HC 10 – 20 ng/mg cf. cholesterol 10 – 20 µg/mg) and it may be in the CNS that the involvement of oxysterols in regulation cholesterol biosynthesis is most significant (Saeed et al., 2014). Another case when oxysterols may be especially important in regulating cholesterol biosynthesis is in response to infection, both bacterial and virus (Bauman, Bitmansour, McDonald, Thompson, Liang & Russell, 2009; Blanc et al., 2013). Rather than bind to SCAP, side-chain oxysterols i.e. 22R-HC, 24S-HC, 25-HC, (25R)26-HC and 24S,25-EC, bind to INSIG and tether the SCAP-SREBP-2 complex in the endoplasmic reticulum (Radhakrishnan, Ikeda, Kwon, Brown & Goldstein, 2007). Elegant experiments performed by Radhakrishnan et al exploiting [3H]25-HC and site-directed mutagenesis confirmed that side-chain oxysterols bind to INSIG via a mechanism involving transmembrane helices 3 and 4, which are also involved in INSIG binding to SCAP (Radhakrishnan, Ikeda, Kwon, Brown & Goldstein, 2007). Radhakrishnan et al reported that 19-HC, 4,4-dimethyl cholesterol and lanosterol did not bind to either INSIG or SCAP and did not inhibit the processing of SREBP-2 (Radhakrishnan, Ikeda, Kwon, Brown & Goldstein, 2007).
Conversely, lanosterol, the first sterol in the cholesterol biosynthesis pathway (Figure 2), stimulates the formation of an INSIG-HMG-CoA reductase (HMGCR) complex (Song, Javitt & DeBose-Boyd, 2005) leading to the ubiquitination and degradation of HMGCR, the rate-limiting enzyme of the cholesterol biosynthesis pathway. Cholesterol has no effect on INSIG-HMGCR formation, however 25-HC and other side-chain oxysterols, e.g. (25R)26-HC, 25H,7O-C and 26-hydroxylanosterol (26-HL, also called 27-hydroxylanosterol) and can induce the formation INSIG-HMGCR complex and reductase ubiquitination (Song, Javitt & DeBose-Boyd, 2005). In an early, and largely forgotten study, Axelson et al showed that (25R)26-HC, (25R)26-hydroxycholest-4-en-3-one ((25R)26-HCO), 7α,(25R)26-dihydroxycholest-4-en-3-one (7α,(25R)26-diHCO), 7α,12α,26-trihydroxycholest-4-en-3-one (7α,12α,26-triHCO) were potent suppressors of HMGCR (Axelson, Larsson, Zhang, Shoda & Sjovall, 1995). The first three of these compounds have been found in brain (Griffiths et al., 2019a; Meljon et al., 2019). However, Axelson et al’s findings were before the involvement of INSIG in HMGCR degradation was uncovered and a mechanism of HMGCR suppression by oxysterols was not described by these authors (Axelson, Larsson, Zhang, Shoda & Sjovall, 1995). Radhakrishnan et al suggested that it is the oxysterol bound form of INSIG can form a complex with HMGCR, just as it can with SCAP, while lanosterol acts by binding to HMGCR, much like cholesterol binds to SCAP (Radhakrishnan, Ikeda, Kwon, Brown & Goldstein, 2007). Lanosterol is a 4,4-dimethyl sterol and a very recent study has confirmed its ability to stimulate HMGCR degradation and its inability to inhibit SREBP-2 cleavage (Chen et al., 2019; Coates & Brown, 2019). Surprisingly, other 4,4-dimethyl sterols in the cholesterol biosynthesis pathway promoted both HMGCR degradation and inhibited SREBP-2 cleavage (Chen et al., 2019).