Molecular Mechanisms of S-adenosyl-L-homocysteine Toxicity

S-adenosyl-L-methionine (AdoMet)-dependent methylation is central to the regulation of many biological processes. At least 50 AdoMet-dependent methyltransferases identified to date methylate a broad spectrum of cellular compounds including nucleic acids, proteins and lipids, and, therefore, play a crucial role in gene expression, signal transduction, and lipid metabolism. Common to all AdoMet-dependent methyltransferase reactions is the release of the strong product inhibitor S- adenosyl-L-homocysteine (AdoHcy), as a by-product of the reaction. Accumulation of AdoHcy is linked to a number of metabolic changes and to numerous diseases. It is currently unclear, which of the multiple AdoMet- dependent methyltransferases are most responsive to AdoHcy alterations and which role dysfunction of individual AdoMet-dependent methylation reactions plays in disease development. We hypothesize that critical methylation reactions exist that become limiting upon AdoHcy-mediated inhibition. Knowledge of AdoHcy-sensitive methylation reactions as well as of cellular processes that are affected by AdoHcy accumulation will greatly improve our understanding of pathological mechanisms involved in AdoHcy-related disorders. Notably, many of the pathological phenotypes that are associated with AdoHcy changes are connected to lipid metabolism. Thus, we propose that AdoHcy-induced inhibition of phospholipid methylation, the major cellular consumer of AdoMet, is one of the key factors in AdoHcy-associated pathology. The observed changes in phospholipid composition and triggering of downstream events like endoplasmic reticulum stress and unfolded protein response as well as accumulation of fatty acids, triacylglycerols and sterols are likely consequences of AdoHcy-mediated inhibition of phospholipid methylation. Using yeast mutant models and mammalian cells as experimental systems, we aim at uncovering at the molecular level the mechanisms leading to deregulated lipid metabolism and impaired ER function under these conditions. We expect that identification and characterization of the critical cellular processes that respond to AdoHcy accumulation will help to understand and explain, and potentially to counteract phenotypes associated with pathological AdoHcy accumulation in humans.

Homocysteine (Hcy) is a non-canonical amino acid that does not occur in proteins. It is produced by demethylation of methionine in the course of transmethylation and either recycled back to methionine via the methylation cycle or converted to cysteine. An accumulation of Hcy in the blood is termed hyperhomocysteinemia (HHcy), the pathological condition, which is present in 5-10% of the general population and up to 30% of the elderly. HHcy is linked to many disorders of modern society including cardiovascular and neurological disease, fatty liver, insulin resistance and cancer. The mechanisms that link HHcy and associated disorders are largely unknown, however evidence suggests that S-adenosyl-L-homocysteine (AdoHcy), a closely related to Hcy metabolite that also accumulates in HHcy, rather than Hcy, is the true trigger of HHcy-associated pathology. Triacylglycerols and phospholipids represent the main energy reservoir and the major membrane constituents, respectively, in the cell. Supplementation of yeast cells with Hcy leads to triacylglycerol accumulation and induces a shift in fatty acid composition of cellular lipids. To understand the role of AdoHcy in these alterations we have established a system that enables dissection of the roles of AdoHcy and Hcy in yeast in vivo. With the help of this system we could show that the accumulation of triacylglycerols and the shift in fatty acid composition of cellular lipids in response to Hcy require conversion of Hcy to AdoHcy and an accumulation of the latter. Our data also show that Hcy affects fatty acid biosynthesis at multiple steps. These includes fatty acid synthase, activity of which is up-regulated, condensing enzymes of fatty acid elongase complex, abundance of which is decreased and localization altered in response to Hcy, as well as fatty acid desaturase. Both impact on fatty acid synthesis, fatty acid elongation as well as fatty acid desaturation appear also to be mediated by AdoHcy in response to Hcy. Impairment of the synthesis of very long chain fatty acids due to interference of AdoHcy/Hcy with fatty acid elongation suggests deregulation of sphingolipid metabolism in yeast treated with Hcy, which as well appear to be mediated by AdoHcy. While AdoHcy is the key trigger of the deregulation of lipid metabolism in response to Hcy, our results also suggest an existence of AdoHcy-independent mechanisms that link elevated Hcy and lipid metabolism. Besides lipid metabolism AdoHcy/Hcy activate in yeast central stress regulatory pathways and interfere with other cellular processes including ribosome biogenesis. Altogether, our data indicate that Hcy mainly via its conversion to AdoHcy elicit profound changes in cell physiology and triggers a regulatory response that involves central regulatory pathways. Understanding of this response will improve our understanding of HHcy-associated pathology and bring closer development of new therapeutic approaches to treat HHcy-associated disorders.