Loss of proteostasis is a hallmark of aging and Alzheimer disease (AD). Here, we identify β-hydroxybutyrate (βHB), a ketone body, as a regulator of protein solubility in the aging brain. βHB is a small molecule metabolite which primarily provides an oxidative substrate for ATP during hypoglycemic conditions, and also regulates other cellular processes through covalent and noncovalent protein interactions. We demonstrate βHB-induced protein insolubility across in vitro, ex vivo, and in vivo mouse systems. This activity is shared by select structurally similar metabolites, is not dependent on covalent protein modification, pH, or solute load, and is observable in mouse brain in vivo after delivery of a ketone ester. Furthermore, this phenotype is selective for pathological proteins such as amyloid-β, and exogenous βHB ameliorates pathology in nematode models of amyloid-β aggregation toxicity. We have generated a comprehensive atlas of the βHB-induced protein insolublome ex vivo and in vivo using mass spectrometry proteomics, and have identified common protein domains within βHB target sequences. Finally, we show enrichment of neurodegeneration-related proteins among βHB targets and the clearance of these targets from mouse brain, likely via βHB-induced autophagy. Overall, these data indicate a new metabolically regulated mechanism of proteostasis relevant to aging and AD.
So why is inducing insolubility a “good thing”? insolubility makes it harder for proteins to participate in toxic reactions, but also makes them harder to get cleared out?
Protein aggregation is a pathological feature of NDDs, but three lines of evidence support an interpretation that metabolite-induced insolubility is ameliorative rather than pathological. First, R-βHB-induced insolubility inhibits amyloid-β cytotoxicity in vitro with a mouse neuronal cell line. Second, in vivo treatment of multiple nematode models of amyloid-β proteoxicity ameliorates their phenotypes. Third, treating mice in vivo with BH-BD revealed clearance, rather than increased accumulation, of the most insoluble fractions of the insolublome, consistent with prior literature.32,45,46 Therefore, our model is that the biochemical mechanism is primed and relatively specific for misfolded proteins, and can interact with much of the proteome at low stoichiometry (misfolded polypeptides comprising a small minority of most proteins at any given time in the cell). As NDD-related proteins are often prone to misfolding, these proteins are overrepresented among R-βHB targets.
We demonstrate that a subchronic treatment with BH-BD as short as two days and up to one week is sufficient to induce a broad shift in the mouse brain insolublome. Our deep proteomic analysis of the one week BH-BD cohort revealed increased insolubilization of NDD-related protein targets in middle insoluble fractions (F2 and F3), and clearance of the most insolubilized aggregates (F4). We additionally found that proteasome-related proteins were significantly enriched among F2 and F3 proteins. This association is consistent with NDD-related protein clearance observed in F4 and may shed light on the potential mechanistic details underlying clearance. We speculate that the UPS degrades proteins in fractions 2 and 3, while the autophagy lysosomal pathway degrades the more insoluble proteins in F4. Protein targets identified ex vivo in both treatment groups were enriched for proteasome and autophagy pathways, suggesting that these protein degradation machineries are key to the proteostatic activities of βHB. Indeed, βHB is a known direct and indirect modulator of autophagic flux.15,54 Further in vivo insolublome investigation demonstrated that a treatment period of only two days may be sufficient in eliciting the same effects as seen during the one week period of treatment, with similar changes in the insolublome. These data lay out the importance of insolubility stratification induced within the brain by R-βHB. Further elucidation of this complex R-βHB insolubilization mechanism will require careful dissection of the affected insolublome.