SIRT3 deacetylase: the Jekyll and Hyde sirtuin

Mitochondrial deacetylase SIRT3 protects against oxidative damage. In an article published online this month in EMBO reports, it is shown to also aggravate paracetamol-induced liver toxicity, calling for caution in trying to pharmacologically enhance SIRT3 activity.EMBO Rep (2011) advance online publication. doi:10.1038/embor.2011.121Post-translational modifications have crucial roles in regulating the functions of many eukaryotic proteins. Among them, lysine acetylation has been traditionally studied in the context of nuclear histone modifications, and was one of the first to be described as part of the ‘histone code'' hypothesis (Kim et al, 2006). More recently, work from several groups has demonstrated that lysine acetylation also modulates the activity of several non-histone proteins. In this context, this modification seems particularly abundant on mitochondrial proteins (Schwer et al, 2009). However, the way in which acetylation influences enzyme function and metabolic reprogramming in pathological states remains unknown. In an article published online this month in EMBO reports, Sack and colleagues shed new light on the role of mitochondrial SIRT3 deacetylase during paracetamol-induced toxicity, describing the mitochondrial protein aldehyde dehydrogenase 2 (ALDH2) as a new target of SIRT3, and a protective role for protein acetylation in this context (Lu et al, 2011).The sirtuin family of NAD⁺-dependent deacetylases comprises seven mammalian homologues (SIRT1–SIRT7) that have diverse functions and cellular localizations (Finkel et al, 2009). Among them, mitochondrial SIRT3 is the main deacetylase involved in the modulation of mitochondrial metabolic and oxidative-stress regulatory pathways (Schwer et al, 2009). SIRT3 seems to mediate protection against oxidative damage under caloric restriction (Someya et al, 2010), as well as promoting enhanced protection against redox and nutrient-excess stress (Zhong & Mostoslavsky, 2011).…these results raise the tantalizing possibility that—at least in the context of [paracetamol] toxicity—the less SIRT3 the betterAcetaminophen (APAP)—commonly known as paracetamol—is a widely used analgesic and anti-pyretic drug that is safe at therapeutic-dose levels. However, APAP overdose has been linked to liver injury in both humans and mice (Jaeschke & Bajt, 2006), with a high mortality rate due to acute liver failure. Remarkably, this hepatotoxic effect seems to be enhanced by fasting (Whitcomb & Block, 1994), a phenomenon that was poorly understood. In the initial phases of cell injury, a product of APAP oxidation—the highly reactive metabolite N-acetyl-p-benzoquinoneimine (NAPQI)—binds to protein cysteine and lysine residues (Zhou et al, 1996), eventually depleting hepatic glutathione and leading to the concomitant hepatotoxicity. Although there has been extensive research, the underlying molecular mechanisms of liver injury have not been fully elucidated.In this new study, Lu and colleagues aimed to decipher the way in which fasting or caloric-restriction exacerbate the redox-stress-dependent toxicity of APAP (Lu et al, 2011). Given the known increase in SIRT3 activity on nutrient deprivation, they proposed that, if protein acetylation inhibits NAPQI binding, SIRT3-mediated deacetylation might aggravate acetaminophen-induced liver injury (AILI).First, they tested whether lack of SIRT3 protects against AILI, by analysing susceptibility to liver injury in SIRT3^+/+ and SIRT3^−/− mice treated with a single toxic dose of APAP under fed and fasted conditions. Strikingly, they found that fasted SIRT3^−/− mice showed less hepatotoxicity than the SIRT3-competent mice. By using two-dimensional gel and immunoblot analyses, they then compared hepatic mitochondrial-protein acetylation profiles between fasted SIRT3^−/− and SIRT3^+/+ mice. In these experiments they identified, among several candidates, ALDH2—a known target of NAPQI, binding to which is known to reduce ALDH2 activity (Landin et al, 1996). This dehydrogenase oxidizes and detoxifies aldehydes—including lipid peroxidation products such as trans-4-hydroxy-2-nonenal (4-HNE; Doorn et al, 2006)—and thus buffers these highly reactive metabolites.…SIRT3 might act as a double-edged sword [raising] a word of caution regarding therapeutic strategies aimed at potentiating SIRT3 activityLu and colleagues then focused on ALDH2. In a series of elegant studies, they demonstrated that ALDH2 is a direct target of SIRT3, and deacetylation of ALDH2 modifies NAPQI binding. Liver mitochondria from SIRT3-deficient mice had increased ALDH2 acetylation, indicating a direct interaction between SIRT3 and ALDH2. ALDH2 was then shown to be a direct target of SIRT3 by using in vitro deacetylation assays. Despite these differences, basal ALDH2 activity remained the same in both genotypes; enzymatic activity was therefore evaluated in response to APAP treatment in fasted mice. Remarkably, SIRT3-deficient mitochondria exhibited approximately 40% higher levels of ALDH2 activity after APAP administration and, consequently, significantly lower levels of 4-HNE adducts were detected, in comparison to SIRT3^+/+ mice. SIRT3 is a known protective factor against oxidative stress; however, these results raise the tantalizing possibility that—at least in the context of APAP toxicity—the less SIRT3 the better.Logically, the next step was to show that the protective effect of SIRT3 deficiency is directly dependent on sustained ALDH2 activity. A marked increased in liver injury in the SIRT3-deficient animals was observed after knockdown of ALDH2 by using a lentiviral short-hairpin RNA approach, supporting their argument. To gain further molecular insight, the authors followed previous observations indicating that binding of NAPQI to ALDH2 diminishes ALDH2 activity (Landin et al, 1996). They hypothesized that SIRT3 might deacetylate ALDH2, in turn increasing its binding to NAPQI and leading to the concomitant inactivation of the protein. Indeed, through elegant SIRT3 gain- and loss-of-function experiments, they demonstrated that SIRT3-dependent deacetylation of ALDH2 enhances binding of the enzyme to NAPQI, whereas SIRT3 inactivation decreases NAPQI binding to ALDH2.The Sack group went one step further and used mass spectrometry to identify ALDH2 Lys 377 as the residue deacetylated by SIRT3. They showed that acetylation of Lys 377 is increased in SIRT3-deficient mice, and a mutant ALDH2 with an acetyl-mimicking mutation (K377Q) exhibited significantly less binding to NAPQI, giving a detailed molecular explanation for the protective effect observed in the absence of this sirtuin.These findings demonstrate that SIRT3-mediated deacetylation of mitochondrial proteins modulates susceptibility to AILI. Furthermore, the identification of ALDH2 as the substrate for SIRT3 deacetylation in this process provides a molecular framework in which to understand the apparent paradox of enhanced APAP toxicity under conditions of fasting or caloric restriction. Fasting induces SIRT3-mediated deacetylation of ALDH2, leading to increased NAPQI binding, which in turn reduces ALDH2 activity. This causes an accumulation of highly reactive adducts, probably contributing to the exacerbated hepatotoxicity observed after APAP treatment under nutrient restriction (Fig 1).Open in a separate windowFigure 1SIRT3-mediated exacerbation of acetaminophen-induced liver injury. SIRT3 deacetylates Lys 377 of ALDH2, making it available for NAPQI binding, which de-activates it. The concomitant reduction in the aldehyde-detoxifying activity of ALDH2 aggravates liver injury. AILI, acetaminophen-induced liver injury; ALDH2, aldehyde dehydrogenase 2; NAPQI, N-acetyl-p-benzoquinoneimine.The toxic effects of AILI have been traditionally addressed by using anti-oxidant therapies based on NAPQI binding to cysteine residues. Surprisingly, the functional outcome of NAPQI binding to lysine residues has not been explored so far, although it was described almost 15 years ago (Zhou et al, 1996). The Sack laboratory approached this issue, providing clear, supportive data for an interesting and provocative hypothesis: although it is widely accepted that SIRT3 has protective, anti-oxidant effects, ALDH2 deacetylation by SIRT3 exacerbates APAP-induced hepatotoxicity. This indicates that SIRT3 might act as a double-edged sword, and raises a word of caution regarding therapeutic strategies aimed at potentiating SIRT3 activity. Although this study provides support for this paradoxical effect, some questions remain. First, is ALDH2 the only SIRT3 substrate involved in this phenotype? The authors show that several other proteins were identified in their study, but their roles remain to be explored. Second, what is the physiological role of SIRT3-mediated ALDH2 deacetylation? Does this modification alter ALDH2 activity under conditions of nutrient stress? If so, how? Third, how general is this phenomenon? Does protein deacetylation modulate the binding of other toxic metabolites to proteins in detoxifying organs, such as the liver? Although answers to these questions await future investigation, one thing is certain: we need to exercise caution when evaluating the therapeutic potential of sirtuin modulators.     

CCN3: Doctor Jekyll and Mister Hyde

CCN proteins are key regulators of signaling pathways that are essential for the control of normal life, from birth to death. As such, they make use of their unique mosaic structure to interact with several other regulatory proteins and ligands that control the fate of living cells. The various functions attributed to the CCN proteins may sometimes appear contradictory, but this situation reflects the complexity of the multimolecular scaffolds in which CCN proteins are engaged and the critical impact of the microenvironment that dictates the bioavailability of the elementary building blocks. CCN3 is one of the best examples of a CCN protein showing biological properties which may at first glance appear opposite or contradictory. Indeed, CCN3 acts both as a tumor suppressor and is associated with higher metastatic potential. Furthermore, the physical interaction of CCN3 with VEGF and its potential antiangionenic activity in glioma cells are in apparent contradiction with its proangiogenic activity in rabbit cornea. In this communication, I am revisiting the observations that led us to these apparent contradictions. After pointing out how the methodologies that were employed might have contributed to the confusion, I briefly discuss the dual biological activities of CCN3 in the context of tumor cell engineering and survival prognosis.     

p27 as Jekyll and Hyde: Regulation of cell cycle and cell motility

p27 is a key regulator of cell proliferation. While it opposes cell cycle progression by binding to and inhibiting cyclin E-Cdk2, T157/198 phosphorylation of p27 promotes its assembly of D-type cyclin-CDKs. In addition to its actions on the cell cycle, p27 regulates CDK-independent cytoplasmic functions. In human cancers, oncogenic activation of the PI3K signaling pathway often results in cytoplasmic mislocalization of p27. Cytoplasmic p27 plays an important role in cell motility and migration; it binds and modulates activation of the RhoA/ROCK cascade. p27:RhoA binding is facilitated by p27 phosphorylation at threonine 198. Accumulation of cytoplasmic p27 leads to increased cellular motility, a critical event in tumor metastasis. Further characterization of post-translational modifications governing p27 localization and its action on RhoA and the actin cytoskeleton may provide critical insights into human cancer metastasis.     

Hydrogen peroxide: a Jekyll and Hyde signalling molecule

Reactive oxygen species (ROS) are a group of molecules produced in the cell through metabolism of oxygen. Endogenous ROS such as hydrogen peroxide (H₂O₂) have long been recognised as destructive molecules. The well-established roles they have in the phagosome and genomic instability has led to the characterisation of these molecules as non-specific agents of destruction. Interestingly, there is a growing body of literature suggesting a less sinister role for this Jekyll and Hyde molecule. It is now evident that at lower physiological levels, H₂O₂ can act as a classical intracellular signalling molecule regulating kinase-driven pathways. The newly discovered biological functions attributed to ROS include proliferation, migration, anoikis, survival and autophagy. Furthermore, recent advances in detection and quantification of ROS-family members have revealed that the diverse functions of ROS can be determined by the subcellular source, location and duration of these molecules within the cell. In light of this confounding paradox, we will examine the factors and circumstances that determine whether H₂O₂ acts in a pro-survival or deleterious manner.     

Acute pancreatitis: proteinase-activated receptor-2 as Dr. Jekyll and Mr. Hyde

"Proteinase-activated" receptor-2 (PAR-2) is a G protein-coupled transmembrane receptor with seven transmembrane domains activated by trypsin. It has been shown in the pancreatic tissue that PAR-2 is involved in duct/acinary cells secretion, arterial tonus regulation and capillary liquid content turnover under physiological conditions. These above mentioned structures play an important role during the development of acute pancreatitis and are profoundly influenced by a high concentration of trypsin enzyme after its secretion into the interstitial tissue from the basolateral aspect of acinar cells. Among the other factors, it is the increase of interstitial trypsin concentration followed rapidly by PAR-2 action on pancreatic vascular smooth muscle cells that initiates ischemic changes in pancreatic parenchyma and that finally leads to necrosis of the pancreas. Consequent reperfusion perpetuates changes leading to the acute pancreatitis development. On the contrary, PAR-2 action on both exocrine and duct structures seems to play locally a protective role during acute pancreatitis development. Moreover, PAR-2 action is not confined to the pancreas but it contributes to the systemic vascular endothelium and immune cell activation that triggers the systemic inflammatory response syndrome (SIRS) contributing to an early high mortality rate in severe disease.     

Dr. Jekyll and Mr. Hyde: A short history of anthrax

The anthrax letters crisis, following the discovery of a major bacterial warfare program in the USSR and the realization that Irak had been on the verge of using anthrax as a weapon during the first Gulf war, had the consequence of putting anthrax back on the agenda of scientists. Fortunately, although it was mostly unknown by the public before these events, it was far from unknown by microbiologists. Already mentioned in the bible as a disease of herbivores, it remained a major cause of death for animals all over the planet until the end of the 19th century, with occasional, sometimes extensive, contamination of human beings. The aetiological agent, Bacillus anthracis, was identified by French and German scientists in the 1860s and 1870s. This was the first time that a disease could be attributed to a specific microorganism. The discovery by Koch that this bacterium formed spores greatly contributed to the understanding of the disease epidemiology. Studies on the pathophysiology of anthrax led to the identification of two major virulence factors, the capsule, protecting the bacilli against phagocytosis, and a tripartite toxin. The latter consists of two toxins with a common component (protecting antigen, PA) that allows the binding to and penetration into cells of two enzymes, the oedema factor EF, a calmodulin dependent adenylate cyclase, and the lethal factor LF, a specific zinc metalloprotease. The primary targets of these toxins would seem to be cells of innate immunity that would otherwise impair multiplication of the bacilli. If detected early enough, B. anthracis infections can be stopped by using antibiotics such as ciprofloxacin. Infection of animals can be prevented by the administration of vaccines, the first of which was developed by Pasteur after an historical testing at Pouilly-le-Fort which marked the beginning of the science of vaccines.     

A Jekyll and Hyde kinase: roles for Cdk5 in brain development and disease

Cyclin-dependent kinase 5 (Cdk5) is a multi-faced kinase implicated in both development and disease of the mammalian central nervous system. These different faces of Cdk5 are preferentially regulated by the activation of Cdk5 by its different binding partners. The precise molecular and cellular mechanisms governing the role of Cdk5 in brain development and disease are unclear. Emerging evidence is now unraveling how Cdk5 normally orchestrates new signaling pathways that dictate the proper maturation and maintenance of the central nervous system. Under pathological conditions, however, Cdk5 activity goes awry and the malevolent face of Cdk5 surfaces. Recently developed animal models that display this deregulated Cdk5 activity reveal the intimate involvement of Cdk5 in tau pathology and neuronal cell death, and underscore the importance of phosphorylation in the progression of neurodegenerative diseases.     

Acanthamoeba differentiation: a two-faced drama of Dr Jekyll and Mr Hyde

The ability of cyst-forming protists such as Acanthamoeba to escape death by transforming into a cyst form, that is resistant to harsh physiological, environmental and pharmacological conditions, has continued to pose a serious challenge to human and animal health. A complete understanding of the fundamental principles of genome evolution and biochemical pathways of cellular differentiation offers unprecedented opportunities to counter detrimental outcomes. Acanthamoeba can elude inhospitable conditions by forming cysts. Here we unravel the processes involved in the phenotypic switching of Acanthamoeba, which are critical in our efforts to find potential targets for chemotherapy.     

Catalytic antibodies: balancing between Dr. Jekyll and Mr. Hyde

The immunoglobulin molecule is a perfect template for the de novo generation of biocatalytic functions. Catalytic antibodies, or abzymes, obtained by the structural mimicking of enzyme active sites have been shown to catalyze numerous chemical reactions. Natural enzyme analogs for some of these reactions have not yet been found or possibly do not exist at all. Nowadays, the dramatic breakthrough in antibody engineering and expression technologies has promoted a considerable expansion of immunoglobulin's medical applications and is offering abzymes a unique chance to become a promising source of high‐precision “catalytic vaccines.” At the same time, the discovery of natural abzymes on the background of autoimmune disease revealed their beneficial and pathogenic roles in the disease progression. Thus, the conflicting Dr. Jekyll and Mr. Hyde protective and destructive essences of catalytic antibodies should be carefully considered in the development of therapeutic abzyme applications.