Ligand-regulated entry into the HRD ERAD pathway—the dark side of allostery

HMG-CoA reductase (HMGR) undergoes regulated degradation as part of feedback control of the sterol pathway. In yeast the stability of the Hmg2 isozyme of HMGR is controlled by the 20 carbon isoprenoid geranylgeranyl pyrophosphate (GGPP): increasing levels of GGPP causes more efficient degradation by the HRD pathway, allowing feedback regulation of HMGR. The HRD pathway is a conserved quality control pathway critical for the ER-associated degradation of misfolded ER proteins. We have explored the action of GGPP in HRD-dependent Hmg2 degradation. GGPP was highly potent as a regulatory molecule in vivo, and in vitro, GGPP altered Hmg2 folding at nanomolar concentrations. These effects of GGPP were absent in a variety of stabilized or non-regulated Hmg2 mutants. Consistent with its high potency, the effects of GGPP were highly specific; other closely related molecules were ineffective in altering Hmg2 structure. In fact, two close GGPP analogues, 2F-GGPP and GGSPP were completely inactive at all concentrations tested, and GGSPP was an antagonist of GGPPs effects in vivo and in vitro. The effects of GGPP on Hmg2 structure and degradation were reversed by chemical chaperones, indicating that GGPP caused selective Hmg2 misfolding. These data indicate that GGPP functions in a manner analogous to an allosteric ligand, causing Hmg2 misfolding through interaction with a reversible, specific binding site. Consistent with this, the Hmg2 protein forms mulitmers. We propose that this “allosteric misfolding,” or mallostery, may be a widely used tactic of biological regulation, with potential for development of small molecule pharmaceuticals that induce selective misfolding.


INTRODUCTION
Protein quality control includes a variety of mechanisms to ensure tolerably low levels of misfolded proteins in the living cell. Among these, selective degradation of misfolded, damaged or un-partnered proteins is often employed for removal of these potentially toxic species. One of the best characterized pathways of degradative quality control is ER-associated degradation (ERAD), entailing a group of ubiquitin-mediated pathways that degrade both lumenal and integral membrane proteins of the endoplasmic reticulum (ER) (1)(2)(3)(4). All degradative quality control pathways show a remarkable juxtaposition in their action. They are all highly specific for misfolded versions of the substrate proteins, yet they recognize a wide variety of distinct and unrelated substrates (5,6). This "broad selectivity" is based on the ability of the ubiquitination enzymes to recognize or respond to specific structural hallmarks of misfolding shared by a wide variety of client substrates. The details and restrictions of these recognition features are still being discovered due to the apparently wide range of ways that E3 ligases can detect their clients (5,(7)(8)(9). We found that indeed GGPP directly influenced the structure of the Hmg2 multispanning anchor, in the low-to-mid nanomolar range. These potent actions of GGPP were highly specific, and in fact were antagonized by a close GGPP analogue both in vivo and in vitro. Furthermore, the effects of GGPP were blocked by a variety of chemical chaperones, indicating that this molecule causes remediable misfolding of the Hmg2 structure to promote HRD recognition. Taken together, these studies lead to a natural model of regulated quality control as a form of allostery that may be widely employed in biology to harness the intrinsic specificity of the many branches of degradative quality control. Because this axis of regulation appears to be based on reversible misfolding due to specific ligand binding, we have given it the name "mallostery" to reflect both the elements of misfolding implied by the prefix, and the action of a selective regulatory ligand that hallmarks allosteric control of many enzymes and other proteins.

Specificity and potency of isoprenoids that
stimulate Hmg2 degradation-In our earlier work, we tested the effects of a variety of sterol pathway molecules on Hmg2 stability (20, 21). We found that only the 20-carbon isoprenoid geranylgeranyl pyrophosphate (GGPP) caused Hmg2 degradation in vivo when added to culture medium (Garza et al., 2009). This surprising ability of exogenous GGPP to stimulate Hmg2 degradation has been a useful feature for study of this regulatory signal (22,23). Because this response is part of a selective negative feedback loop, we posited that the GGPP signal would be specific, physiologically relevant, and highly potent. To more systematically evaluate these ideas, we first performed dose response experiments on pathway isoprenoids alone and in combination.
We examined the effects of candidate isoprenoids on Hmg2 stability in vivo using flow cytometry on cells expressing Hmg2-GFP, which undergoes regulated degradation identical to the native enzyme (24), but provides no additional enzymatic contribution to signal production. Each was tested at a variety of concentrations by direct addition to yeast cultures, followed by a 1 hour incubation and flow cytometry. GGPP caused Hmg2-GFP degradation at culture concentrations as low as 1 µM, reaching a maximum at approximately 20 µM ( Fig 1C). The effect of GGPP on Hmg2-GFP was highly specific: the 15 carbon farnesyl pyrophosphate (FPP) and the non-phosphorylated 20 carbon geranylgeraniol (GGOH) had no effect in vivo. Similarly, neither of the earlier pathway isoprenoids, isopentenyl pyrophosphate (IPP) or geranyl pyrophosphate (GPP), had any effect on Hmg2-GFP at concentrations up to 27 µM.
When microsomes isolated from cells expressing myc L -Hmg2-GFP are treated with a low concentration of trypsin, immunoblotting the protected myc epitope after SDS-PAGE reveals a characteristic time-dependent pattern of proteolyzed fragment production ( Fig 2B).
Because the myc tag is protected, the total myc immunoblotting signal intensity remains unchanged. We developed this assay to explore how signals from the sterol pathway affect the structure of Hmg2 to render it more susceptible to the HRD quality control pathway (26). In those early studies we found that the rate of myc L -Hmg2-GFP proteolysis was altered by manipulations that affect the in vivo stability of the protein, such that in vitro proteolysis occurred more rapidly when microsomes were prepared from strains where the degradation signals are high (26). In vivo, Hmg2 or Hmg2-GFP is strongly stabilized by chemical chaperones (29). Similarly, proteolysis of microsomal myc L -Hmg2-GFP is drastically slowed by addition of the chemical chaperone glycerol, and this structural change is fully reversible (25). We employed this in vitro structural assay to explore the possibility that sterol pathway signals directly affected the structure of Hmg2 to allow regulated degradation.
In those studies, we showed that the 15 carbon neutral isoprenoid farnesol (FOH) caused significant acceleration of in vitro myc L -Hmg2-GFP trypsinolysis, again preserving the cleavage pattern but altering the kinetics (26). This effect of FOH is fully reversible. Furthermore, mutants of Hmg2-GFP that do not respond to in vivo degradation signals, including a substitution of a small number of amino acids known as "TYFSA", or a single point S215A point mutant of a highly conserved residue of the sterol-sensing domain (SSD), do not respond to farnesol in the limited proteolysis assay (22,26). Although those results were intriguing and biologically appropriate, the biological role of farnesol per se was unclear.
Although there was a clear structure-activity relationship for farnesol in the proteolysis assay, the concentrations required to cause the in vitro effects were very high (EC 50 ~ 100 uM), and farnesol is extremely toxic to yeast. In the times since these studies, we discovered that the bona fide physiological regulator was the normally made isoprenoid GGPP, which also causes the structural transition of Hmg2-GFP in the proteolysis assay (20). Accordingly we returned to this assay to evaluate the specificity and potency of GGPP in a more controlled setting.
In striking contrast to FOH, we found that GGPP was a potent modifier of Hmg2 structure. GGPP accelerated in vitro trypsinolysis at concentrations as low as ~15 nM, with an apparent half-maximum concentration lower than 200 nM (Fig 2C left, 2E).
Intriguingly, this concentration is in the range of the K M of yeast enzymes that use GGPP as a substrate (30-32), indicating that this concentration is likely physiologically relevant since the enzymes are "tuned" to concentrations of substrate that exist in their milieu. The maximal effect of GGPP was similar to that seen with the largest effects of FOH reported earlier, about a 5 fold increase in proteolysis rate. The highly stable mutant S215A, which does not respond to FOH in the in vitro assay, also did not respond to GGPP at any concentration tested (Fig 2C, right). Hmg2-GFP again became more susceptible to proteolysis, and to the same extent as the original exposure ( Fig 2G).

Antagonism of GGPP action in vitro and in vivo-
The GGPP analogues 2F-GGPP and GGSPP had no ability to stimulate Hmg2-GFP degradation in vivo ( Fig     Hmg2 forms multimeric structures (Fig 6A).
When only Hmg2-GFP or 1myc-Hmg2 was expressed in a strain, we were unable to detect the other tag in input lysates or immunoprecipitations ( Fig 6A).
We asked if GGPP could affect Hmg2 to target pharmacologically, and have in fact been referred to as the "undruggable proteome" (56).
The UPS system has already been tapped as a tool for pharmacological targeting of these undruggable protein through regulated degradation.
Two main strategies have emerged so far: targeting proteins directly to specific E3 ligases, such as VHL (57), and targeting proteins with ligands fused to a long hydrophobic molecule, or "greasy patch," to mimic a misfolded protein (45).
Directing proteins specifically to quality control by cleaver discovery of mallosteric regulators that cause selective unfolding may offer another approach for targeting the undruggable proteome, and one that nature has clearly already discovered during evolution. Yeast strains and plasmids-Yeast strains (Table 1) and plasmids (Table 2)   Middle set was treated with GGPP then washed, and the right group was treated with GGPP, washed, then re-treated with GGPP. All samples were then subjected to limited proteolysis assay.

Reagents-Geranylgeranyl
Note re-addition of GGPP gave precisely the same response as first addition.