In vivo model for adenine accumulation and toxicity
Purine biosynthesis pathways are crucial for the normal function of cells and are conserved between yeast and humans. Both the adenine phosphoribosyltransferase (APRT) and adenosine deaminase (ADA) enzymes take part in adenine salvage in humans and mutations in their encoding genes can lead to the accumulation of adenine and its derivatives22,23. Mutation in APRT leads to the APRT deficiency and mutation in ADA leads to ADA deficiency. In budding yeast, the APRT and ADA orthologs (APT1 and AAH1, respectively) are similarly involved in adenine salvage33. Apt1 catalyzes the formation of AMP from adenine and Aah1 converts adenine to hypoxanthine. To reveal the importance of these two enzymes for cell growth, we generated an adenine salvage mutant by disruption of both APT1 and AAH1 genes. The double mutant showed a slow growth phenotype on synthetic defined (SD) medium containing a specific mixture of amino acids and nucleobases (SD complete), compared to the growth of the wild-type and both single mutants (Fig. 1a, b). Interestingly, removal of adenine from the medium dramatically improved cell growth of the metabolite salvage mutant and had no significant effect on any of the other strains (Fig. 1a, b). The double mutant showed a slow growth phenotype also when using a minimal medium containing glycerol as a nonfermentable carbon source (Supplementary Fig. 1). Thus, the reduced growth of the salvage mutant depended only on the presence of adenine, regardless of the carbon source, ruling out the possibility that the salvage mutant had lost respiratory competence due to mitochondrial mutations. To verify that the toxic effect observed for the salvage mutant is indeed due to disruption of the AAH1 and APT1 genes, single copy plasmids carrying both genes were introduced to the mutant cells by transformation. As shown in Supplementary Fig. 2, the restoration of the genes indeed rescued the growth phenotype.
Altogether, the presence of adenine at the normal concentration used for wild-type yeast growth leads to a significant cell growth decrease in a strain that is defective in the biosynthesis downstream to adenine. This is indeed an unusual phenomenon as in most cases (excluding mutations in transport systems) the absence of a given metabolite, rather than its presence in the physiological concentration needed for normal yeast growth, serves as a limiting factor of wild-type yeast growth34. This is analogous to the toxicity of normal metabolite concentrations observed in some inborn error of metabolism disorders, such as PKU and tyrosinemia35, where the RDA for the general population is actually toxic to the affected individuals, due to accumulation of the metabolites in the absence of salvage. The effect indeed appears to reflect toxicity, as increasing adenine levels in the medium led to decreased cell growth in a dose-dependent manner (Fig. 1c, d), similar to the toxic effect previously observed in in vitro cell culture studies4. It should be noted that the toxic effect on cell growth due to adenine accumulation is very similar to the outcome of protein amyloid expression in yeast cells26,28,29.
Non-linear response to adenine feeding
The dose–response curve of cell growth as a function of adenine concentration was fitted using a four-parameter logistic equation (4PL), producing a typical-sigmoidal shaped curve, with no effect at lower concentrations and a sharp increase in the inhibitory effect upon reaching a critical concentration threshold (Fig. 1d). Thus, cell growth appears to be affected by adenine levels in a non-linear cooperative manner. This is consistent with the mechanism of nucleation-growth as observed in micelle formation (assembly above a critical micelle concentration, CMC) or the formation of amyloids by the assembly of protein monomers36,37,38. Indeed, in vitro studies also showed non-linear self-assembly of adenine at different concentrations (Supplementary Fig. 3 and Supplementary Methods). Thus, we present in vitro as well as in vivo data regarding adenine accumulation at different concentrations and the possible correlation between the assembly mechanism of fibril formation and cellular growth. Our results imply that above a critical concentration, the favorable energetic state dictates the formation of the toxic assemblies, while at lower concentrations the metabolites are most likely at the normal physiological state.
To quantify the intracellular concentrations of adenine, gas chromatography mass-spectrometry (GC–MS) was used. Analysis of the cellular adenine concentrations under different conditions indicated that on SD media in the presence of the metabolite, adenine levels were indeed significantly higher in the salvage mutant as compared to the wild-type strain (×45 fold; Fig. 1e), suggesting a clear correlation between the feeding amounts of adenine and growth inhibition. Similarly, when not following a very strict diet, inborn error of metabolism patients have 1–2 orders of magnitude higher concentrations of metabolites, as compared to the general population10.
Visualization of adenine accumulation by Raman imaging
To detect the accumulation of adenine in vivo, we utilized Raman microspectroscopy. Raman spectroscopy coupled with microscopy has recently emerged as a promising tool to trace intracellular processes in vivo and was successfully used in yeast to follow glucose assimilation into intracellular components39. Raman spectrum provides rich and highly specific chemical information and thus intracellular molecular distribution can be visualized at a sub-μm spatial resolution by Raman microspectroscopy. Moreover, being a vibrational spectroscopic technique, label-free imaging could be performed, as it requires no exogenous dye probe. Space-resolved Raman spectra obtained at three different points in salvage mutant single cells (aah1Δ apt1Δ) in the presence of adenine showed Raman bands characteristic to proteins (Fig. 2a, c). Major features included 1004 cm−1 [phenylalanine ring breathing], 1250 cm−1 [amide III], 1340 and 1448 cm−1 [C–H bend], and 1655 cm−1 [amide I]40,41,42. By carefully examining the region between 800 and 700 cm−1, we could observe a band at 785 cm−1 corresponding to ring breathing modes of nucleobases such as uracil, cytosine, thymine, and nucleic acid backbone vibration (Fig. 2b). Additionally, we also observed a band at 724 cm−1 corresponding to adenine ring breathing modes (Raman band of adenine in solid and solution forms are shown in Supplementary Fig. 4). According to these observations, we chose the 785 cm−1 band as a marker of nucleic acids in general while 724 cm−1 served as an adenine marker43,44,45. As expected, the intensities of these two bands varied depending on the location in the cell. As shown in Fig. 2b, while the red and black spectra showed intense adenine marker with very low nucleic acid band (indicating high prevalence of adenine), respectively, the spectrum in green showed comparable intensities of these two bands, which is typical of nucleic acids. Thus, these bands can be used to study adenine accumulation.
In order to further investigate adenine accumulation in living yeast cells, we performed Raman imaging experiments on both mutant and wild-type yeast in the presence (Fig. 2d) or absence (Fig. 2e) of adenine. We first imaged macromolecules such as proteins, using phenylalanine ring breathing mode at 1004 cm−1 (magenta). No significant difference was observed, indicating that proteins were similarly distributed under all conditions. In the presence of externally added adenine, images constructed using 724 cm−1 showed high intracellular abundance of adenine in the double mutants, while its distribution was very low in wild-type yeast (blue). In the absence of externally added adenine, the intensity of intracellular adenine was comparable in both mutant and wild-type cells. To exclude the contribution of adenine from nucleic acids, we calculated the relative intensity of 724 cm−1 images (blue) and nucleic acids 785 cm−1 images (green) (724/785 cm−1), as shown in red. Relative intensity images showed adenine accumulation and the appearance of subcellular regions of high adenine concentration only in double mutant yeast cells in the presence of adenine. Average intracellular intensity of 724 cm−1 per pixel, 785 cm−1 per pixel, and the relative intensity per pixel of WT and aah1Δapt1Δ cells were calculated (Fig. 2f). The results reinforce the imaging results, showing significant adenine levels only in the mutant cells.
Amyloid-like assemblies formation upon adenine accumulation
To examine whether the observed non-linear behavior of the dose-dependent toxicity is indeed associated with self-organization of the metabolites into amyloid-like assemblies in vivo, the cells were stained with ProteoStat, an amyloid-specific fluorescent dye. This reagent was previously shown to facilitate specific and sensitive detection of amyloid aggregates in living cells46. To validate the possible identification of amyloid fibrils by ProteoStat staining in yeast, detection of the prion protein Sup35 was examined (Supplementary Fig. 5), showing significant and gradual increase in the staining of [psi−] compared to two types of [PSI+] aggregates, weak [PSI+] and strong [PSI+]. Flow cytometry and confocal microscopy were then employed to detect the presence of intracellular amyloid-like adenine aggregates. Both techniques clearly indicated the presence of amyloid-like structures in the adenine salvage mutant. Flow cytometry showed a higher degree of aggregation in the mutant compared to wild-type cells in the presence of the metabolite (Fig. 3a). Moreover, consistent with the indicated adenine sensitivity (Fig. 1a), removal of adenine from the medium reduced the level of aggregation in the mutant cells (Fig. 3a). Confocal microscopy further allowed the identification of the intracellular localization of the adenine aggregates, showing clearly stained dots only in the salvage mutant and specifically following the addition of adenine (Fig. 3b). Z-stack followed by 3D reconstruction (Fig. 3c), as well as projection of a single section of the Z-stack (Fig. 3d), showed that the stained dots were localized inside the cell, excluding the possibility of their attachment to the outer membrane or cell wall. Furthermore, Hoechst staining showed no co-localization, ruling out the possibility that the stained dots were inside the nucleus (Fig. 3e).
The Hsp104 chaperon was previously shown in yeast to play a pivotal role in the formation of numerous aggregates by structurally unrelated proteins and its deletion partially restored the viability of cells expressing Aβ and polyQ in yeast models for Alzheimer’s disease and Huntington’s disease, respectively47,48. To test whether the toxicity following adenine accumulation in the salvage mutant is mediated by Hsp104, cell growth upon adenine addition was examined in the presence of guanidine hydrochloride that was repeatedly shown to inhibit Hsp104 activity, as well as in a hsp104Δ mutant background (Supplementary Fig. 6a, b). No Hsp104 dependency was observed, suggesting that the toxicity induced by adenine accumulation involves a different mechanism than the Hsp104-associated one. To further validate this observation, the chaperon response was examined using a Hsp104-mCherry strain49 (Supplementary Fig. 6c). While Hsp104 recruitment and accumulation was observed under heat shock stress and in the presence of adenine both in wild-type and in the salvage mutant cells, no such dots appeared under optimal growth temperature, suggesting a different mechanism other than the Hsp104-associated one that was previously reported.
In vivo formation of adenine amyloid-like assemblies
To confirm that the staining with the ProteoStat dye specifically identifies adenine amyloid-like structures, antibodies against adenine fibril structures were generated, as we have previously described for phenylalanine and tyrosine fibrils3,50. After staining with the anti-adenine fibril antibodies, fluorescent dots could be detected only in the salvage mutant in the presence of adenine (Fig. 3f). This result suggests that the observed cellular toxicity is directly linked to self-assembled amyloid-like adenine structures.
Inhibition of adenine toxicity by TA
We next aimed to manipulate the formation of the intracellular adenine amyloid-like structures formed at high cellular concentrations of adenine upon feeding. Polyphenols comprise a large group of natural and synthetic small molecules, which were repeatedly shown to inhibit the formation of protein amyloid fibrils51,52, including the formation of aggregates that is associated with neurodegenerative diseases53. We examined the effect of TA, a widely studied polyphenol which was suggested as a potent inhibitor of β-amyloid fibrillation and of the assembly of the PrPsc prion protein54,55. Moreover, we have recently demonstrated the inhibition of adenine self-assembly in vitro by this polyphenol compound15. We found that in the presence of adenine, the addition of TA to the yeast media clearly improved cell growth of the salvage mutant in a dose-dependent manner (Fig. 4a, b). Staining of the cells with the ProteoStat amyloid-specific dye followed by flow cytometry allowed the detection of a decrease in metabolite aggregation in the presence of the inhibitor, despite the external addition of adenine to the media (Fig. 4c). Furthermore, the absence of the stained dots following the addition of TA was demonstrated by confocal microscopy (Fig. 4d). Based on mass-spectroscopy analysis (Supplementary Fig. 7a, b and Supplementary Methods), TA and adenine were detected in yeast cell debris as well as inside the cells, in yeast soluble material, indicating that TA enters the cells. To verify the molecular identity of the TA peak, we performed the same analysis on samples that contained adenine but not TA, showing only the peak corresponding to adenine (Supplementary Fig. 7c, d).
In order to ascertain that the inhibition by TA represents a general phenomenon applicable for other known amyloid inhibitors, baicalein, an additional polyphenolic inhibitor of protein amyloid formation, that was recently shown to bear therapeutic potential for Alzheimer’s and Parkinson’s disease56 was examined. Indeed, the use of this inhibitor resulted in a significant effect on yeast cell growth (Supplementary Fig. 8a, b) and on adenine aggregation (Supplementary Fig. 8c), as well as in a dose-dependent inhibition of adenine self-assembly in vitro (Supplementary Fig. 8d).
TA mechanism of action
In order to examine the in vivo mechanism underlying the inhibition of adenine amyloid-like formation by TA, the inhibitor was added at different time points of yeast growth. TA was found to hinder the formation of adenine amyloid-like assemblies when added at earlier time points of yeast growth, while it had no effect on their growth when added at later stages, suggesting that TA is most effective at the nucleation and early oligomerization stage of the metabolite self-assembly (Fig. 5a). These results further support a correlation between adenine aggregation into ordered structures and growth inhibition, suggesting that the toxic effect is induced by adenine accumulation into amyloid-like species. This effect can be rescued by either removal of adenine from the medium or addition of the amyloid inhibitor, thereby modulating the assembly process.
Finally, we examined whether the dramatic effect of the inhibitor on the viability of the mutant cells actually reflects a change in the concentration of adenine in the metabolite salvage model, in addition to the inhibition of amyloid-like structure formation. For this purpose, the intracellular concentrations of adenine with and without the inhibitor were compared using mass spectrometry. Evidently, while the addition of the inhibitor drastically improved cell growth, the intracellular levels of adenine remained constant (Fig. 5b). Thus, the change in cell growth appears to occur specifically due to the inhibition of adenine aggregate formation, and not as a result of a decrease in adenine levels, ruling out the possibility of an indirect effect of TA on adenine availability in the medium. This result, in addition to studies demonstrating the ability of TA to inhibit amyloid-like fibrillation of adenine in vitro15, further implies that the assemblies, rather than free metabolite molecules, mediate cell toxicity (as modeled in Fig. 5c).