Mallosteric Regulation of the Sterol Pathway

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HMG-CoA reductase (HMGR) is a key enzyme of the sterol pathway. HMGR undergoes feedback- regulated degradation conserved from yeast to humans. We exploit this conservation to understand the machinery and mechanisms at play in regulated degradation of the yeast Hmg2 isozyme. Degradation occurs by the HRD ER-associated degradation (ERAD) quality control pathway, also responsible for the degradation of misfolded ER proteins. Hmg2 ERAD is regulated by sterol pathway molecule GGPP. GGPP accomplishes control by causing reversible misfolding that triggers HRD pathway degradation. GGPP’s action on Hmg2 has many features of allosteric control; we have named this type of regulation “mallostery” to combine the ideas of misfolding and allostery. In the proposed studies we will unravel the mallosteric regulation of Hmg2, to better understand sterol pathway control, and for the high potential mallostery holds as broadly applicable avenue of drug discovery. Specifically, we will : 1) Study the features of Hmg2 that allow GGPP-dependent reversible misfolding: GGPP’s effect on the Hmg2 transmembrane regions is highly specific, and requires the broadly conserved sterol sensing domain (SSD). We will discover the sequence features of Hmg2 responsible for mallosteric control by GGPP, investigating the role of known Hmg2 motifs and discovering sequence features with unbiased genetic approaches; 2) Explore the mechanism of GGPP dependent regulation of Hmg2- We will test the hypothesis that GGPP is a high potency ligand for Hmg2, causing reversible misfolding through development of direct interaction assays, analysis of GGPP analogues, and through in vitro reconstitution of regulated ubiquitination of Hmg2 using a number tools and approaches developed in the last funding cycle; and 3) Discover the role of INSIG proteins in GGPP-mediated misfolding of Hmg2- The INSIG proteins are critical in mammalian lipid homeostasis, and are conserved in yeast (Nsg1 and 2). As in mammals, yeast INSIGs function by transducing sterol signals. Yeast INSIG controls GGPP-dependent Hmg2 misfolding in a manner dependent on the biosynthetic sterol lanosterol. Thus the yeast INSIGs impose a second layer of regulation on mallosteric regulation by GGPP. We will use the tools, mutants, and methods from both the previous funding period, and the above studies to explore the mechanism and physiology INSIG function in Hmg2 regulated stability. We will also execute a genetic screen to glean the broader biology of INSIGs, about which almost nothing is known despite the key and highly conserved roles of these proteins in human lipid homeostasis and pathology.