Prion propagation and inositol polyphosphates

Abstract

The [PSI+] prion is a folded in-register parallel β-sheet amyloid (filamentous polymer) of Sup35p, a subunit of the translation termination factor. Our searches for anti-prion systems led to our finding that certain soluble inositol polyphosphates (IPs) are important for the propagation of the [PSI+] prion. The IPs affect a wide range of processes, including mRNA export, telomere length, phosphate and polyphosphate metabolism, energy regulation, transcription and translation. We found that 5-diphosphoinositol tetra(or penta)kisphosphate or inositol hexakisphosphate could support [PSI+] prion propagation, and 1-diphosphoinositol pentakisphosphate appears to inhibit the process.

Yeast prions were originally viewed as a model for the mammalian transmissible spongiform encephalopathies, particularly the Mad Cow disease epidemic in the UK at that time (Wickner 1994). Now there is increasing evidence that most of the common human amyloidoses have prion-like aspects, and in some cases may be frankly infectious (Jaunmuktane, et al. 2015, Junker and Walker 2011, Kraus, et al. 2013), so the importance of the yeast model is even greater.

While nearly 10 yeast and fungal prions have been described (reviewed in (Wickner, et al. 2015)), the [PSI+] and [URE3] prions, self-propagating amyloids of Sup35p and Ure2p (the latter a regulator of nitrogen catabolism) have been most intensively investigated. The folded in-register parallel β-sheet architecture, shown for several, can explain how these amyloids can self-template their structures, and so how they can act as genes composed of protein, with multiple alleles (called prion variants in yeast or prion strains in mammals) (Fig. 1, (Gorkovskiy, et al. 2014, Shewmaker, et al. 2006, Wickner, et al. 2015).

Fig. 1.

Conformational templating mechanism based on the folded in-register parallel β-sheet architecture of yeast prion amyloids (Wickner, et al. 2015). Favorable interactions between identical amino acid side chains (hydrogen bonds or hydrophobic interactions) maintain the in-register architecture, and drive a monomer joining the end of an amyloid filament to acquire turns/folds at the same locations as those of molecules already in the filament. This conformational templating, analogous to DNA’s sequence templating, allows these proteins to be genes (Wickner, et al. 2015). Different prion variants (with the same protein sequence) are hypothesized to have the folds/turns at different locations.

Overproduction or deficiency of many chaperones and other cell components have been found to result in prion loss (e.g. (Chernoff, et al. 1995, Higurashi, et al. 2008, Kryndushkin, et al. 2008, Sharma and Masison 2008, Troisi, et al. 2015). Deletion or overexpression of a protein can efficiently eliminate a prion by very indirect means, or, although direct, by producing a condition that does not occur in the wild. But recently we have proposed a new ‘definition’ of anti-prion system/component, namely a protein that eliminates a prion when expressed at its normal level. With this approach several anti-prion systems have been shown, starting with proteins whose curing activity was first shown by overproduction. Hsp104 overproduction cures the [PSI+] prion (Chernoff, et al. 1995), and Btn2p or Cur1p overproduction cure the [URE3] prion (Kryndushkin, et al. 2008). But most of the prion variants isolated in strains lacking these curing activities are cured by simply restoring the normal level of the active protein (Gorkovskiy, et al. 2017, Wickner, et al. 2014). In effect, what has been discovered is a large array of [PSI+] and [URE3] variants that would normally never appear because the normal levels of the Hsp104 or Btn2p or Cur1p would cure them as they arise.

In a general screen for anti-prion systems using the yeast knockout collection, an siw14Δ mutation was found to allow many variants of [PSI+] to propagate that could not propagate in SIW14⁺ cells (Wickner, et al. 2017). However, overexpression of Siw14p did not cure any [PSI+] variants isolated in normal cells. Since Siw14p was recently identified as a pyrophosphatase specific for the 5-pyrophosphate-inositol pentakisphosphate (5PP-IP₅, see Fig. 2) (Steidle, et al. 2016), it was suspected that some inositol poly/pyro-phosphates promote prion propagation (Wickner, et al. 2017). Indeed, arg82 mutants, that lack most of the soluble inositol poly/pyro-phosphate species are unable to propagate the large majority of [PSI+] variants. Genetically engineered blockage of various points in the IP pathway led to the conclusion that either IP₆ or 5PP-IP₄ are certainly sufficient for [PSI+] propagation, and presumably 5PP-IP₅ can also fulfill this function (Fig. 2). The requirement of [PSI+] for the IPs is not eliminated by removing the [PSI+]-curing activity of Hsp104, and the [URE3] prion does not have such an IP requirement (Wickner, et al. 2017).

Fig. 2.

Biosynthetic pathways of the soluble inositol poly/pyro-phosphates (IPs) (reviewed in (Saiardi, et al. 2017, Tsui and York 2010). IP₆ and 5PP-IP₄, and probably 5PP-IP₅, are capable of helping [PSI+] propagate (green arrows), and 1PP-IP₅ may inhibit [PSI+] propagation (red blockage symbol) (Wickner, et al. 2017).

Inositol poly/pyro-phosphates are known to have a wide array of functions (Table 1), but none of these functions immediately explain their requirement for [PSI+] prion propagation. The need for inositol pyrophosphates for the ‘environmental stress response’, was the most suggestive, but the ability of kcs1Δ vip1Δ mutants (that make no inositol pyrophosphates (Mulugu, et al. 2007)) to support [PSI+] indicates that the prion is supported by another IP function (Wickner, et al. 2017). These kcs1Δ vip1Δ mutants do make inositol hexakisphosphate (IP₆), but kcs1Δ ipk1Δ mutants do not make IP₆ and do not support [PSI+], implicating IP₆ as another species capable of helping [PSI+] propagate (Wickner, et al. 2017). In spite of being able to make IP₆, kcs1Δ ddp1Δ mutants (see Fig. 2) lose [PSI+]. These mutants make elevated levels of 1-disphosphoinositol pentakisphosphate (Mulugu, et al. 2007), suggesting that this compound may inhibit [PSI+] propagation (Wickner, et al. 2017).

Table 1.

Some of the Functions of inositol poly/pyro-phosphates.

Function	Reference
Endocytic trafficking	(Saiardi, et al. 2002)
Environmental stress response, histone deacetylation	(Dubois, et al. 2002, Worley, et al. 2013)
RNA editing	(Macbeth, et al. 2005)
Translation termination	(Bolger, et al. 2008)
Phosphate assimilation	(Lee, et al. 2007)
mRNA export	(York, et al. 1999)
Chemotaxis	(Luo, et al. 2003)
Telomere length	(York, et al. 2005)
Transcription	(Odom, et al. 2000)
Chromatin remodeling	(Shen, et al. 2003, Steger, et al. 2003)

These experiments have revealed an unexpected level of control over prion propagation. However, the mechanism by which these inositol poly/pyro-phosphates exert their positive and negative control is unknown. The inositol pyrophosphates contain high energy pyrophosphate bonds and have been shown able to pyrophosphorylate proteins at certain amino acid residues that are already monophosphorylated (Saiardi, et al. 2004). It is conceivable that this activity is involved in supporting [PSI+] by direct modification of Sup35p, but the ability of IP₆, lacking pyrophosphates, to support [PSI+] argues against this idea. IPs are very highly charged molecules and can bind to positively charged regions on certain proteins (e.g., (Macbeth, et al. 2005)), but the prion domain of Sup35p, the part that actually forms the amyloid structure, has very few charged residues, including only two arginines and one lysine in the 123 residues.

It is quite possible that the IPs are acting on some other protein to augment, diminish or qualitatively change its activity in such a way that [PSI+] propagation is allowed. Solid resins with IP₆ or 5PP-IP₅ covalently attached were designed and used to purify proteins from S. cerevisiae that had specific affinity for one of these molecules (Wu, et al. 2016). Note that these are two of the molecules that we found were each sufficient to support [PSI+] propagation. Among the ~40 proteins that bound to both resins were Ssb1 and Ssb2, ribosome – associated Hsp70 chaperones; Hsp26p, a small heat shock protein; and Sse1p, an Hsp70 family member with nucleotide exchange activity. The Ssb proteins (Chernoff and Kiktev 2016, Chernoff, et al. 1999) and Sse1p (Fan, et al. 2007, Kryndushkin and Wickner 2007) are known to affect [PSI+] generation and propagation, but preliminary efforts to prove these as targets of the IPs have not yet succeeded. Whatever the mechanism, Siw14p appears to be working by limiting the levels of certain inositol pyrophosphates, preventing the propagation of a group of [PSI+] variants that need a high level of these compounds.

This work opens a new avenue for investigation of anti-prion/anti-amyloid modalities. Do human amyloidoses involve IPs in some way? The IP biosynthetic pathways in yeast and humans are very similar, and most pathogenic human amyloids studied have, like yeast prions, the folded in-register parallel β-sheet architecture (reviewed by (Tycko 2014, Tycko and Wickner 2013). Of course, even [URE3] is not affected by IP deficiency, so there need not be direct carry-over of these results to mammals. Nonetheless, these results again highlight the familiar theme that studies of yeast, rapidly and cheaply carried out, can form the basis for understanding of human disease processes.