Mechanistic insight into the mitochondrial acetylome

More than 200 types of post-translational modifications have been discovered within proteomes, creating a complex landscape of protein diversity and function. PTMs alter a protein’s charge or structure in a way that can affect activity, localization and interactions with other cellular components.
 
One class of PTMs includes the addition of acyl groups derived from Coenzyme-A pools (e.g., acetyl-CoA and succinyl-CoA) to the epsilon-amino group of lysine residues. Two papers published recently in The Journal of Biological Chemistry lend new insights into both enzymatic and nonenzymatic mechanisms for lysine acetylation/acylation of mitochondrial proteins.
 
Lysine acetylation was first discovered as a mechanism that regulates histone function, but it is now known as a common PTM that is highly conserved across the evolution of prokaryotes and eukaryotes. More recently, succinyl-CoA and other thioester molecules (e.g., malonyl-CoA) also have been found to modify lysine residues, a modification generally referred to as lysine acylation. In the mitochondria, hyperacetylation of metabolic enzymes has been implicated in a number of human illnesses, including diabetes, obesity and cancer, which are related to metabolic syndrome and mitochondrial dysfunction.
 
While it is known that hyperacetylation is induced by metabolic stress like starvation or from the loss of deacetylase activity, researchers have limited knowledge about the presence of enzymatic lysine acetyltransferases, or KATs, and alternate acetylation pathways within the mitochondria.
 
In the first JBC paper, Krista L. Stilger and William J. Sullivan Jr. at the Indiana University School of Medicine discovered the presence of an Elp3 homologue, TgElp3 — the catalytic subunit of the elongator complex — in the genome of the single-celled parasite Toxoplasma gondii when searching for potential KAT gene candidates.
 
They conducted an initial study with recombinant TgElp3 and demonstrated KAT activity in vitro. Based on bioinformatic analyses, the TgElp3 gene and homologous genes from select members of the Apicomplexa phylum are unique in composition, containing a C-terminal transmembrane domain that is missing from other eukaryotic Elp3 proteins.
 
The researchers used a variety of imaging techniques to show that TgElp3 is localized to the outer mitochondrial membrane, with its C-terminal tail anchored there and the catalytic domain jutting into the cellular cytosol. This orientation suggests the protein may acetylate proteins in the cytosol, on the outer mitochondrial membrane surface, and as they are being transported into the mitochondria (Figure 1).

Figure from Stilger and Sullivan
Figure 1: Stilger and Sullivan demonstrate with immunoelectron microscopy that a recombinant Elp3-homologue (black dots) in Toxoplasma gondii localizes to the outer mitochondrial membrane (M), where it may acetylate proteins that are transported into the mitochondria.

In higher eukaryotes, the elongator complex possesses intrinsic acetyltransferase activity and acts on substrates that include histone H3 and alpha-tubulin, affecting both transcriptional elongation in the nucleus and cell division activities in the cytosol. Mutations in Elp3 and other complex subunits of higher eukaryotes have been implicated in a number of nervous-system and developmental disorders.
 
Because all other elongator complex subunits appear to be missing in the T. gondii genome, the authors propose that the TgElp3 protein may have evolved initially to function as a general KAT protein for early-branching eukaryotes. However, some literature also suggests that Elp3 has been detected within the mitochondria of the HeLa human cell line.
 
Therefore, the researchers also performed a fractionation study with mouse brains and showed evidence that a truncated version of Elp3 protein localizes inside the mitochondria, although the transport mechanism is unknown. Future studies that focus on the role of Elp3 in regulation of the mitochondrial acetylome and identification of its transport mechanism to the mitochondria in higher eukaryotes are needed.
 
The second JBC paper, which was classified by the journal’s editors as a “Paper of the Week,” was by Gregory R. Wagner and R. Mark Payne, also of IUSM. Wagner and Payne propose a nonenzymatic mechanism for lysine acylation in the mitochondria based on the chemically catalytic conditions that naturally exist within this organelle (Figure 2).

Figure from Wagner and Payne
Figure 2: Wagner and Payne propose a base-catalyzed nucleophilic acyl substitution reaction for the nonenzymatic acetylation of lysine in the mitochondrial matrix, which is facilitated by the physiological conditions of the mitochondria (i.e., high pH and acyl-CoA levels).

To test their hypothesis, the researchers simulated mitochondrial conditions in vivo (i.e., elevated pH plus high concentrations of acetyl-CoA or succinyl-CoA) with mouse liver mitochondrial extracts and employed antibody staining to detect acyl-lysine formation. The results clearly demonstrated that both thioesters nonenzymatically modify lysine residues.
 
Further evidence for a nonenzymatic mechanism included experiments that show lysine acylation of both denatured mitochondrial extracts and a nonmitochondrial protein and lysine acetylation activity in the presence of CoA, a known inhibitor of acetyltransferases.
 
Lysine acylation in the mitochondria is regulated by the action of the NAD(+)-dependent sirtuins, SIRT3 and SIRT5, which act as deacylases to remove acetyl and succinyl groups, respectively. In this study, a recombinant SIRT3 was shown to reduce the levels of acetyl-lysine residues on the chemically modified mitochondrial proteins. The researchers propose that SIRT3 and SIRT5 may have evolved exclusively to regulate nonenzymatic lysine acylation in the mitochondria.
 
These results do not rule out the action of acetyltransferases (or other acyltransferases) in the mitochondria. However, this paper underscores that the physiological role of such enzymes is unclear, because hyperacetylation within the mitochondria leads to detrimental metabolic effects and acetylation events can proceed nonenzymatically.
 
These two studies shed light on a long-standing mystery that has surrounded mitochondrial protein acetylation, offering two mechanisms that are not mutually exclusive. Both enzymatic and nonenzymatic lysine modifications may be taking place at the mitochondria, providing cells multiple options to maintain mitochondrial homeostasis.

Donna KridelbaughDonna Kridelbaugh (@science_mentor) is a science communications consultant and founder of the Science Mentor blog project. She offers a variety of image consulting and marketing services for early career researchers. Learn more at http://sciencementor.wordpress.com.

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