Proteostatic Tactics in the Strategy of Sterol Regulation

Abstract

In eukaryotes, the synthesis and uptake of sterols undergo stringent multivalent regulation. Both individual enzymes and transcriptional networks are controlled to meet changing needs of the many sterol pathway products. Regulation is tailored by evolution to match regulatory constraints, which can be very different in distinct species. Nevertheless, a broadly conserved feature of many aspects of sterol regulation is employment of proteostasis mechanisms to bring about control of individual proteins. Proteostasis is the set of processes that maintain homeostasis of a dynamic proteome. Proteostasis includes protein quality control pathways for the detection, and then the correction or destruction, of the many misfolded proteins that arise as an unavoidable feature of protein-based life. Protein quality control displays not only the remarkable breadth needed to manage the wide variety of client molecules, but also extreme specificity toward the misfolded variants of a given protein. These features are amenable to evolutionary usurpation as a means to regulate proteins, and this approach has been used in sterol regulation. We describe both well-trod and less familiar versions of the interface between proteostasis and sterol regulation and suggest some underlying ideas with broad biological and clinical applicability.

Keywords: ERAD; HMG-CoA reductase; Hrd1; INSIG; SCAP; SSD.

Figure 1

The sterol or mevalonate pathway. Only the salient features of this widely conserved and essential biosynthetic pathway are shown. The carbon number for some intermediates is shown in black circles (because carbon is black). Orange arrows indicate the early, oxygen-independent section of the pathway. Green arrows indicate the later, sterol-synthesizing, oxygen-dependent section of the pathway.

Figure 2

Eukaryotic tactics of sterol pathway regulation. A highly stylized cartoon showing the identities and basic features of the proteins involved in control of the sterol pathway. (a) Mammalian regulated ER retention of SCAP (and bound SREBP). (b) Mammalian regulated ER-associated degradation (ERAD) of HMG-CoA reductase (HMGR). (c) Saccharomyces cerevisiae regulated ERAD of the Hmg2 HMGR isozyme. (d) Schizosaccharomyces pombe regulated ER retention of SCAP (and bound SREBP). Sterol-sensing domain proteins are shown in green, INSIGs in orange, SREBP cargo in purple, and the ER membrane in yellow (the lumen is below, and the cytosol is above). Ligands are ball shaped. GGPP denotes geranylgeranyl pyrophosphate.

Figure 3

Geranylgeranyl pyrophosphate (GGPP)-mediated reversible misfolding of Hmg2. (a) An example of the trypsinolysis assay used to explore Hmg2-GFP structure. The time course of the trypsinolysis using a high concentration of trypsin, measured by SDS-PAGE and epitope tag blotting. The dark fragment in the 1-min lane is the initial product generated by rapid removal of GFP at the start of the assay. Hmg2-GFP with a luminal epitope is also depicted. (b) Effect of added GGPP on the rate of Hmg2 cleavage in the limiting trypsinolysis assay. (c) Concentration dependence of GGPP in altering Hmg2-GFP structure in the trypsinolysis assay, along with several other isoprenoids. GGPP EC₅₀ ∼ 50 nM. Extent of proteolysis is defined as the fraction of the major fragment degraded. Abbreviations: FOH, farnesol; FPP, farnesyl pyrophosphate; GGOH, geranylgeraniol. Adapted from Wangeline & Hampton (2017). (d) The GGPP effect is highly specific; 2F-GGPP has no effect on Hmg2 trypsinolysis, whereas GGSPP is an antagonist of GGPP in vitro and in vivo.

Figure 4

Mallostery, or a reversible, ligand-mediated misfolding model for sterol-sensing domain (SSD) proteins, in which the SSD motif imparts the ability to undergo reversible misfolding. We posit that the SSD protein functions as a multimer and effects a variant of allosteric regulation, allowing for ligand-mediated misfolding as a regulatory tactic.