The identification of a Sir2-related enzyme in the mammalian mitochondrion raises a number of interesting questions related to the NAD-dependent enzymatic activity associated with this family of enzymes and the pivotal role played by NAD in mitochondrial metabolism. In almost every respect, hSIRT3 behaves as a classical mitochondrial matrix protein. Its dependency on an NH2
-terminal cleavable presequence has been reported for other mitochondrial matrix proteins (Roise et al., 1986
; von Heijne et al., 1989
; Abe et al., 2000
). Mitochondrial targeting sequences are characterized by the presence of positively charged and hydrophobic residues (negative charged residues are very rare) (Roise et al., 1988
) and tend to adopt a helical, frequently amphipathic, conformation. Mutational analysis of an amphipathic helix within the NH2
terminus of hSIRT3 showed that eliminating the positive charges by substituting arginines with glycines or glutamines led to a loss of mitochondrial import. Disrupting the putative helical conformation of the targeting signal by introducing proline residues within the helix also suppressed import despite the conservation of three charged residues. Thus, both the net charge and the helical nature of the targeting region are necessary for import of hSIRT3 into mitochondria. Whereas the NH2
terminus of hSIRT3 is critical for mitochondrial import, preliminary experiments indicate that a fusion protein consisting of the NH2
-terminal 25 amino acids of hSIRT3 and GFP is not targeted to mitochondria. This result indicates that other domains of hSIRT3, most likely the first NH2
-terminal 100 amino acids are involved in its mitochondrial import.
The hSIRT3 cDNA predicts a protein product of ~43.6 kD; however, an antiserum specific for the COOH terminus of hSIRT3 detected a smaller form of 28 kD as the major form in cultured cells. A product of similar size was observed as the major protein after transfection of hSIRT3 with COOH-terminal FLAG tag. Both experiments are consistent with the deletion of a leader sequence at the NH2 terminus of hSIRT3. The observation that a deletion mutant of hSIRT3 lacking the first 25 amino acids was not targeted to the mitochondria and was also not processed to a smaller product suggests either that mitochondrial targeting is necessary for proteolytic processing or that targeting of the protease to hSIRT3 is dependent on its first 25 amino acids.
Most proteins with NH2
-terminal mitochondrial targeting sequences are processed after import into the mitochondrial matrix by MPP (Arretz et al., 1991
). MPP processed hSIRT3 in vitro to a new product similar in size to the 28-kD processed endogenous hSIRT3 protein, suggesting that MPP is responsible for hSIRT3 cleavage within the mitochondrial matrix. Mutational analysis of hSIRT3 revealed that arginines 99 and 100 are necessary for cleavage by MPP in vitro. The presence of critical arginine residues within the cleavage site recognized by MPP has been described previously (Ogishima et al., 1995
; Song et al., 1996
; Shimokata et al., 1997
). Although these mutations completely suppressed cleavage by MPP in vitro, we observed residual cleavage of the same mutant (hSIRT3-R99/100G) in vivo after transfection. These results are likely to reflect the limiting digestion conditions imposed by our in vitro assay or, less likely, the presence of an alternative mitochondrial processing enzyme. In addition, transfection of hSIRT3/R99–100G into mammalian cell led to the appearance of novel cleavage products, suggesting additional processing events. Interestingly, 30% of mitochondrial precursor proteins processed by MPP are further processed by the mitochondrial intermediate peptidase (MIP) (Kalousek et al., 1988
; Isaya et al., 1991
). MIP cleavage removes an additional octapeptide from the NH2
terminus of the MPP-processed precursor. It is possible that cleavage by MIP or another mitochondrial protease is responsible for the processing of hSIRT3 when MPP processing has been inhibited. Finally, we noted in our alkaline fractionation experiments that the incompletely processed form of hSIRT3 was associated with the membrane fraction, indicating that entry of hSIRT3 into the matrix compartment is likely to represent a critical step in its proteolytic processing, consistent with the matrix localization of MPP.
We had initially observed that hSIRT3 expressed in E. coli or after in vitro translation systems was catalytically inactive. Remarkably, MPP cleavage of hSIRT3 synthesized in vitro resulted in its catalytic activation. This was not observed with a catalytically inactive hSIRT3-H248Y, although this mutant protein was cleaved to the same extent as wild-type hSIRT3. This observation indicates that enzymatic activation of hSIRT3 by proteolytic processing by MPP is not an artifact linked to the MPP preparation but is strictly dependent on the intrinsic enzymatic activity of hSIRT3. This control also indicates indirectly that enzymatic activity of hSIRT3 is not necessary for its proteolytic processing.
These observations suggest that hSIRT3 is synthesized as an inactive precursor within the cytoplasm, transported to the mitochondrial matrix, where it is proteolytically processed to activate its enzymatic potential. This model would allow the safe transfer of a latent enzyme and its selective activation when the proper destination in the mitochondrial matrix has been reached. While our in vitro evidence strongly supports this model, we have had difficulties evaluating the role of hSIRT3 cleavage on its enzymatic activity in vivo. When the mutant hSIRT3-R99/100G harboring the mutated MPP recognition site was transfected into cells, cleavage efficiency was reduced, but a certain amount of processed hSIRT3 protein could still be immunoprecipitated and exhibited NAD-dependent deacetylase activity. Therefore, whether uncleaved full-length hSIRT3 can exert NAD-dependent deacetylation in vivo remains an unanswered question. However, our experimental evidence indicates that the processed form of hSIRT3, either endogenous or after transfection, is indeed enzymatically active.
Although we have demonstrated that hSIRT3 can deacetylate a histone H4 peptide, mitochondria lack histone proteins. Therefore, other nonhistone mitochondrial proteins are likely to be substrate(s) of hSIRT3. Two recent reports have described the specific deacetylation of the transcription factor p53 by another human Sir2 homologue protein, hSIRT1 (Luo et al., 2001
; Vaziri et al., 2001
). We have also obtained evidence that a third human Sir2 protein, hSIRT2, is predominantly cytoplasmic (unpublished data). These observations are in agreement with previous reports that several mammalian Sir2 homologues are found in nonnuclear subcellular localizations (Zemzoumi et al., 1998
; Afshar and Murnane, 1999
; Yang et al., 2000
; Perrod et al., 2001
). As an alternative, hSIRT3 could also target a nonprotein substrate for deacetylation.
The selective targeting of a mammalian Sir2-like protein to mitochondria is intriguing because mitochondria represent the bioenergetic and metabolic centers of eukaryotic cells. It had been speculated that the phylogenetically conserved family of Sir2 proteins is involved in sensing cellular energy and redox states (Smith et al., 2000
). Given its mitochondrial matrix localization, hSIRT3 might differ from other Sir2 proteins in its sensitivity to metabolic activity. In contrast to the cytoplasm where NAD levels can change in response to ATP abundance, the mitochondrial matrix content of NAD is believed to be stable and not subject to changes caused by varying ATP levels (Vinogradov et al., 1972
; Devin et al., 1997
; Tischler et al., 1977
; Di Lisa et al., 2001
). The presence of stable NAD levels might ensure the constitutive activity of hSIRT3 in the mitochondrial matrix leading to the constitutive deacetylation of one or several mitochondrial proteins.
In contrast to class I and II HDACs, the deacetylation reaction catalyzed by Sir2-like proteins does not lead to the production of acetate. Rather, Sir2-like enzymes catalyze a unique reaction in which the cleavage of NAD and the deacetylation of substrate are coupled with the formation of O-acetyl-ADP-ribose, a novel metabolite (Tanner et al., 2000
; Borra et al., 2002
; Jackson and Denu, 2002
). This metabolite has intrinsic biological activity and causes a delay/block in oocyte maturation (Borra et al., 2002
). These observations imply the existence of cellular enzymes that can efficiently utilize O-acetyl-ADP-ribose. We will explore the possibility that hSIRT3 functions as an NAD-dependent sensor and controls a variety of metabolic activities through the formation of O-acetyl-ADP-ribose.
In addition, certain conditions leading to an abrupt decrease in mitochondrial NAD might shut off or severely decrease the activity of hSIRT3, leading to a relative hyperacetylation of its substrates and to a decrease in O-acetyl-ADP-ribose production. These conditions include the opening of the mitochondrial permeability transition pore (mtPTP), a high-conductance channel within the inner mitochondrial membrane. Adding calcium to isolated mitochondria leads to a rapid depletion of mitochondrial NAD, most likely due to the opening of mtPTP (Vinogradov et al., 1972
). NAD released from the matrix is hydrolyzed by NAD glycohydrolases (NADases) in the intermembrane space. Hydrolysis of NAD by NADases leads to the formation of ADP-ribose and nicotinamide, itself an inhibitor of hSIRT3 enzymatic activity. Reactive oxygen species (ROS) can also trigger mtPTP opening and NADase stimulation (Bernardi, 1999
; Bernardi et al., 1999
; Ziegler et al., 1997
). Interestingly, as we show in this article, disruption of Δψm inhibits mitochondrial import of hSIRT3, thereby reducing its mitochondrial content. Therefore, mtPTP opening caused by apoptotic stimuli, ROS or calcium elevation is likely to inhibit hSIRT3 function by several independent mechanisms including matrix NAD depletion, increased NAD hydrolysis, formation of nicotinamide and inhibition of hSIRT3 import into the matrix. According to this model, constitutive hSIRT3 activity could play an important role in protection against apoptosis, and its inhibition might lead to increased acetylation of factors directly involved in apoptotic pathways.
HSIRT3 could also play a pathogenic role in cancer. The gene encoding hSIRT3 maps to chromosome 11p15.5, a region close to the telomere subjected to genomic imprinting. This region contains a major yet unidentified tumor suppressor gene (Henry et al., 1991
; Weksberg et al., 1993
; for review see Feinberg, 2000
) and a locus associated with the Beckwith-Wiedemann syndrome (BWS), which causes prenatal overgrowth and predisposition to cancer (Lee et al., 1999
The exact function of hSIRT3 remains to be elucidated. However, the localization of this enzyme to the mitochondrial matrix already gives important clues to its potential substrates and biological functions. Future experiments will address these important questions.