N-terminal acetylation and the N-end rule pathway control degradation of the lipid droplet protein PLIN2

Abstract

Perilipin 2 (PLIN2) is a major lipid droplet (LD)-associated protein that regulates intracellular lipid homeostasis and LD formation. Under lipid-deprived conditions, the LD-unbound (free) form of PLIN2 is eliminated in the cytosol by an as yet unknown ubiquitin (Ub)-proteasome pathway that is associated with the N-terminal or near N-terminal residues of the protein.Here, using HeLa, HEK293T, and HepG2 human cell lines, cycloheximide chase, in vivo ubiquitylation, split-Ub yeast twohybrid, and chemical cross-linking-based reciprocal co-immunoprecipitation assays, we found that TEB4 (MARCH6), an E3 Ub ligase and recognition component of the Ac/N-end rule pathway, directly targets the N-terminal acetyl moiety of N␣-terminally acetylated PLIN2 for its polyubiquitylation and degradation by the 26S proteasome. We also show that the TEB4-mediated Ac/N-end rule pathway reduces intracellular LD accumulation by degrading PLIN2. Collectively, these findings identify PLIN2 as a substrate of the Ac/N-end rule pathway and indicate a previously unappreciated role of the Ac/N-end rule pathway in LD metabolism.

N-terminal methionine excision of proteins creates tertiary destabilizing N-degrons of the Arg/N-end rule pathway

“…Likewise, in naa20⌬ cells, the ablation of the NatB-mediated Ac/N-end rule pathway would rapidly trigger the other NME/ NatB-mediated Arg/N-end rule pathway, thus exhibiting the observed comparable degradation of Deg1 fusions in both WT cells and naa20⌬ cells (49), which remains to be tested. Furthermore, our most recent genetic and biochemical experiments reveal that TEB4 (a mammalian homolog of Doa10) (23) more preferentially binds to Nt-acetylatable native proteins, such as RGS2 and PLIN2, than to their nonacetylatable counterparts, which is in agreement with our previous identification of TEB4 (Doa10) as an Ac/N-recognin (26).…”

“…The Ac/N-end rule pathway has been implicated in the regulation of protein subunit stoichiometry, G-protein signaling-mediated blood pressure, circadian rhythms, plant stress responses, lipid droplet maintenance, etc. (13,(22)(23)(24)(25)(26).…”

Cholesterol increases protein levels of the E3 ligase MARCH6 and thereby stimulates protein degradation

“…D2 is a deiodinase that activates thyroid hormone by converting the pro-hormone T4 to T3, the biologically active thyroid hormone (12), and RGS2 is a regulator of G proteins that lowers blood pressure by decreasing signaling through G␣ q (13). The other established substrates of MARCH6 are itself, a mutant bile salt exporter pump (14), mutant NPC1 (7), the perilipin PLIN2 (8), and the cholesterol synthesis enzymes SM and HMGCR (4). Cholesterol synthesis is especially energy-intensive (45), and interestingly, bile acids induce energy expenditure by promoting intracellular thyroid hormone activation via induction of D2 (46).…”

“…MARCH6 is also one of several E3 ligases implicated in the degradation of HMGCR, another rate-limiting cholesterol synthesis enzyme (4 -6). A mutant form of the cholesterol-trafficking protein, NPC1, is targeted to the proteasome by MARCH6-catalyzed ubiquitination (7), and a protein involved in lipid-droplet formation, PLIN2, is also targeted by MARCH6 (8). MARCH6 additionally affects transcriptional regulation in cholesterol homeostasis, ultimately reducing expression of the inducibledegrader of LDL receptor (IDOL) (9), another E3 ligase, which degrades lipoprotein receptors.…”

The cholesterol synthesis enzyme lanosterol 14α-demethylase is post-translationally regulated by the E3 ubiquitin ligase MARCH6

“…Possible human candidates for LDM include the E3 ubiquitin ligases Hrd1 and membrane-associated ring-CH-type finger 6 (MARCH6) as they target cholesterol synthesis enzymes [31,32], and gp78 and CHIP as they target other cytochrome P450s for degradation [33]. MARCH6 is involved in the degradation of SM and HMGCR [31], other sterol and lipid metabolism substrates [34][35][36], and is itself stabilised by cholesterol [37], indicating an important role in cholesterol homeostasis.…”