Increasing evidence suggest that specific metabolic alterations associated with cancer cells may not be ancillary to their transformation but instrumental to their tumorigenic potential by mediating cell proliferation, growth and survival (Vander Heiden et al., 2009
). Many oncogenes and tumor suppressor genes known to promote excess cell proliferation also alter biosynthetic (or anabolic) processes. For example, Akt expression stimulates glucose uptake and glycolysis, the pentose phosphate pathway and fatty acid synthesis. c
-Myc expression promotes glutamine metabolism as well as purine and pyrimidine biosynthesis. Furthermore, mutations in genes encoding metabolic enzymes have been identified by cancer genetic association studies (Vander Heiden et al., 2009
). However, how specific metabolites contribute to increased proliferation and apoptotic resistance in tumor cells remains a central unanswered question.
The proto-oncogene Bcl-xL has a prominent role in promoting cell survival and cancer development (Boise et al., 1993
). It is well-established that Bcl-xL protects against apoptosis by directly binding and inhibiting Bax/Bak-oligomerization mediated mitochondrial permeabilization. However, certain Bcl-xL mutants, such as F131V/D133A and G148E, that are unable to bind to Bax or Bak, nevertheless retain 70–80% anti-apoptotic activity of WT Bcl-xL (Cheng et al., 1996
). Curiously, Bcl-xL has also been shown to regulate mitochondrial respiration and metabolism (Gottlieb et al., 2000
; Vander Heiden et al., 1999
). Whether the metabolic function of Bcl-xL contributes to its role in mediating apoptotic resistance is unclear.
Our unexpected identification of an N-terminal acetyltransferase, Arrest Defective 1 (dArd1) in a genome-wide RNAi screen in Drosophila
cells for apoptotic regulators (Yi et al., 2007
) prompted us to posit that protein N-alpha-acetylation, a major N-terminal modification, links cell metabolism to apoptotic induction in cancer cells. Since dARD1 is epistatic to Diap1, a direct inhibitor of caspases in Drosophila
, and ARD1 is required for caspase activation in mammalian cells (Yi et al., 2007
), the role for ARD1 in mediating caspase activation is evolutionarily conserved. How ARD1 regulates caspase activation has not yet been illustrated.
In mammalian cells, protein N-alpha-acetylation is mediated by the highly conserved N-acetyltransferase protein complexes (NatA, NatB, NatC, NatD, and NatE). The NatA complex consists of the catalytic subunit, Arrest Defective 1 (hNaa10p/ARD1), and the auxiliary subunit, N-acetyltransferase 1 (NAT1/hNaa15p/NATH); whereas NatB consists of N-terminal acetyltransferase 3 (hNaa20p/NAT3) and mitochondrial distribution and morphology 20 (hNaa25p/Mdm20). Although the Nat complexes are implicated in regulating cell cycle progression, cell proliferation and tumorigenesis, the mechanisms that connect N-alphaacetylation to the cellular protein apparatus are unknown (Ametzazurra et al., 2008
; Polevoda and Sherman, 2003
; Starheim et al., 2008
; Starheim et al., 2009
). Recent N-acetylome studies reveal incomplete acetylation status of proteins (Arnesen et al., 2008
; Goetze et al., 2009
). Although a commonly accepted view is that partial acetylation results from degenerate nature of protein N-terminal sequences, we considered the possibility that protein N-alpha-acetylation might be regulated, an alternative hypothesis that had not been tested due to technical limitations.
Here we developed a novel biochemical approach to assess the status of endogenous levels of protein N-alpha-acetylation. Using this assay, we show that protein N-alpha-acetylation levels are sensitive to alterations in metabolism and Bcl-xL expression. Bcl-xL overexpression leads to reduced levels of acetyl-CoA and hypoacetylation of protein N-termini through a Bax/Bak independent mechanism. Conversely, Bcl-xL−/− mouse embryonic fibroblasts show increased levels of acetyl-CoA as well as protein N-alpha-acetylation levels. Protein N-alpha-acetylation deficiency in Bcl-xL overexpressing cells contributes to apoptotic resistance since increasing acetyl-CoA production can rescue this deficiency in protein N-alpha-acetylation and sensitize Bcl-xL cells to cell death. Our study suggests that regulation of acetyl-CoA availability and protein N-alpha-acetylation may provide a Bax/Bak independent mechanism for Bcl-xL to regulate apoptotic sensitivity.