Biogerontology DOI 10.1007/s10522-015-9591-y
REVIEW ARTICLE
Drosophila melanogaster mitochondrial Hsp22: a role in resistance to oxidative stress, aging and the mitochondrial unfolding protein response Genevie`ve Morrow . Marie Le Pe´cheur . Robert M. Tanguay
Received: 13 March 2015 / Accepted: 1 July 2015 Ó Springer Science+Business Media Dordrecht 2015
Abstract Aging is characterized by the accumulation of dysfunctional mitochondria. Since these organelles are involved in many important cellular processes, different mechanisms exist to maintain their integrity. Among them is the mitochondrial unfolding protein response, which triggers the expression of a set of proteins aimed at re-establishing mitochondrial homeostasis. The induction of mitochondrial chaperones expression, particularly of Hsp60 and Hsp70, is a hallmark of this pathway. In Drosophila melanogaster, Hsp22 is also up-regulated by mitochondrial stress. This small heat shock protein is one of the members of the family to be localized inside mitochondria. One characteristic of Drosophila Hsp22 is its preferential up-regulation during aging and in oxidative stress conditions. It is a beneficial protein since its over-expression increases lifespan and resistance to stress while its down-regulation is detrimental. This review focuses on Drosophila Hsp22 and its links with the mitochondrial unfolding protein response and the aging process, in addition to highlight the important role of this sHSP in mitochondrial homeostasis. G. Morrow M. Le Pe´cheur R. M. Tanguay (&) Laboratoire de Ge´ne´tique Cellulaire et De´veloppementale, De´partement de biologie mole´culaire, biochimie me´dicale et pathologie, Institute de Biologie Inte´grative et des Syste`mes (IBIS) and PROTEO, Universite´ Laval, Pavillon CE-Marchand, 1030 avenue de la me´decine, Que´bec, QC G1V 0A6, Canada e-mail: [email protected]
Keywords Hsp22 Small heat shock protein (sHSP) Oxidative stress Reactive oxygen species (ROS) Mitochondrial unfolding protein response (UPRMT) Aging
Introduction Aging is characterized by a decline of cellular functions that ultimately leads to death. The molecular mechanisms underlying this process are not all understood and multiple theories have been proposed to explain it [reviewed in (Vina et al. 2007; Zhou et al. 2011)]. Among them the mitochondrial free radical theory of aging has been intensively studied in the past decades and places mitochondria at the center of the aging process. According to this theory, reactive oxygen species (ROS) produced as by-products of mitochondrial ATP synthesis are causing damages to DNA, lipids and proteins that eventually lead to cellular dysfunction and death. This theory has been questioned recently due mainly to the failure of antioxidant therapies to extend aging and to the existence of organisms like the naked mole rat which have low levels of antioxidants and high levels of oxidative damage but nevertheless live seven times longer than the domestic mouse [reviewed in (Scialo et al. 2013)]. At physiological levels, it is now admitted that ROS signal information through oxidation of specific sulfhydryl groups on key signaling proteins (Genova
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and Lenaz 2015; Orr et al. 2013; Wang et al. 2012). However there is a threshold above which ROS become harmful and induce oxidative stress (Dai et al. 2014; Genova and Lenaz 2015; Orr et al. 2013; Rigoulet et al. 2011). Mitochondria are the main generators of ROS, and complex I is considered as the major source of ROS involved in aging (Barja 2014; Genova and Lenaz 2015; Holzerova and Prokisch 2015; Orr et al. 2013; Scialo et al. 2013). Indeed, longlived animals have species-specific low mitochondrial ROS generation rates at complex I, and the extension of longevity by caloric restriction can be mimicked by inhibition of this ETC complex [reviewed in (Barja 2014; Genova and Lenaz 2015; Ristow and Schmeisser 2011, 2014; Scialo et al. 2013)]. Interestingly, a recent study in Caenorhabditis elegans has shown that mitochondrial ROS can increase longevity in presence of a detrimental level of cytoplasmic ROS, demonstrating that ROS have a compartment specific effect on lifespan (Schaar et al. 2015). Mitochondria are essential organelles that are involved in multiple cellular processes. In addition to producing ATP through oxidative phosphorylation, they are central executioner of apoptosis and involved in calcium homeostasis/storage as well as in multiple anabolic and catabolic processes such as fatty acid ßoxidation, branch-chain amino-acid metabolism, heme and hormone synthesis and ammonia detoxification. Therefore, mitochondria have to adapt to the cell needs and are in constant communication with the nucleus (Haynes et al. 2007; Haynes and Ron 2010; Runkel et al. 2014; Schieke and Finkel 2006). While dysfunctions of mitochondria have been reported in numerous diseases as well as in aging, up-regulation of the mitochondrial stress response has gained recognition as a potential pro-longevity mechanism [reviewed in (Dai et al. 2014; Hill and Van Remmen 2014; Ziegler et al. 2015)]. This specific stress response includes changes in mitochondrial dynamic (fission/fusion), regulation of mitochondrial biogenesis/turnover, retrograde signaling and mitochondrial unfolding protein response (UPRMT) [reviewed in (Hamon et al. 2015; Hill and Van Remmen 2014; Ikeda et al. 2015; Jensen and Jasper 2014; Vilchez et al. 2014)]. The UPRMT is a mitochondrial-to-nuclear signal transduction pathway initiated by the accumulation of unfolded proteins in mitochondria and resulting in the induction of stress response proteins such as anti-
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oxidants, chaperones and proteases. It has first been observed in C. elegans but is now recognized in other eukaryotes although it is not as well defined (Haynes et al. 2007; Haynes and Ron 2010; Hill and Van Remmen 2014; Jensen and Jasper 2014). In C. elegans, mitochondrial unfolded proteins are cleaved by caseinolytic protease P (ClpP), an ATPdependent mitochondrial matrix protease. The resulting peptides are then exported out of the mitochondria to the cytosol by HAF-1 (an ATP binding cassette transporter) where they weaken mitochondrial import, thereby preventing ATFS-1 (activating transcription factor associated with stress 1) transcription factor to be imported into the mitochondria and degraded by the Lon protease. Consequently, ATFS-1 translocates to the nucleus and activates mitochondrial stress response genes in cooperation with ubiquitin-like 5 (UBL-5) and DVE-1 (an homeodomain-containing transcription factor) to ultimately reconstitute mitochondrial homeostasis (Aldridge et al. 2007; Cristina et al. 2009; Haynes et al. 2007; Haynes and Ron 2010; Haynes et al. 2010; Jovaisaite et al. 2014; Nargund et al. 2012). Among mitochondrial chaperones, Hsp60/Hsp10 and mitochondrial Hsp70 (mtHsp70) have been associated with UPRMT in different organisms. In Drosophila melanogaster, two other mitochondrial chaperones are involved in this important stress response, namely Hsp22 and tumor necrosis factor receptor associated protein 1 (TRAP1) (Baqri et al. 2014; Shen and Tower 2013; Tower 2014; Tower et al. 2014). While TRAP1 is a member of the HSP90 family of chaperone, Hsp22 is a small heat shock protein (sHSP) that is preferentially up-regulated during aging. We will here review the evidence linking D. melanogaster Hsp22 to flies UPRMT and aging processes.
Hsp22 is a mitochondrial chaperone induced by mitochondrial stress In D. melanogaster there are 12 sHSPs, which have distinctive developmental expression pattern and intracellular localization [reviewed in (Morrow and Tanguay 2015)]. Among them, Hsp22 is the only one to be found in mitochondria. More precisely it is located in the mitochondrial matrix although a small fraction has been shown to sediment with
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A Survival following 48 hours of paraquat exposure (%)
mitochondrial membrane proteins especially after stress (Morrow et al. 2000). As most members of the sHSP family, Hsp22 prevents in vitro heat-induced protein aggregation and maintains heat-denatured protein in a refoldable state for the ATP-dependent chaperones such as Hsp60 and Hsp70 (Morrow et al. 2006). In vivo, its chaperone activity has not been assayed directly, but as other sHSPs it has been shown to interact with proteins involved in a wide range of cellular functions suggesting that its chaperone function is not restricted to specific clients (Basha et al. 2004; Giot et al. 2003; Guruharsha et al. 2011; Haslbeck and Vierling 2015). During development, Hsp22 is expressed in the course of metamorphosis from larvae to pupae and is up-regulated during aging (King and Tower 1999; Michaud et al. 2002). Importantly, Hsp22 is also robustly up-regulated upon mitochondrial stress. Indeed, hsp22 is one of the most up-regulated genes in tko25 flies that have an insufficient mitochondrial translational capacity due to a mutation in mitoribosomal protein S12 (mRpS12) (Fernandez-Ayala et al. 2010). Moreover, hsp22 together with l(2)efl (another sHSP from Drosophila), are the sHSPs genes showing the greatest induction upon oxidative stress (H2O2, paraquat, C99.5 % O2 atmosphere (Girardot et al. 2006; Gruenewald et al. 2009; Hirano et al. 2012; Landis et al. 2004, 2012). Reagents that induce ROS production are recognized activator of UPRMT, together with interventions that deplete the mitochondrial genome, delete mitochondrial electron transport chain (ETC) genes and inhibit mitochondrial protease activity (Aldridge et al. 2007; Copeland et al. 2009; Durieux et al. 2011; Haynes and Ron 2010; Jensen and Jasper 2014; Yoneda et al. 2004). Paraquat is a methyl viologen that generates superoxide through ETC complex I inhibition. While it is detrimental at high dose it has been shown to increase lifespan at low dose, highlighting the potential of ROS as signaling molecules [reviewed in (Ristow and Schmeisser 2011, 2014)]. Interestingly, hsp22 expression is induced following both robust and mild oxidative stresses brought by paraquat (Hirano et al. 2012). At the protein level, Hsp22 is up-regulated in presence of 1 mM paraquat, a dose which do not significantly alter fly survival (Fig. 1). Of note is the concomitant but very mild induction of Hsp60 in the same conditions suggesting that both heat shock proteins (HSPs) can be up-
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Fig. 1 Differential induction of mitochondrial chaperones upon oxidative stress mediated by paraquat. Five days-old W1118 flies (60–80/condition) were starved (no food) for 6 h and then put in presence of a piece of Whatman 3 M filter soaked with 200 ll of 1 % sucrose (control, 0) or different concentrations of paraquat diluted in 1 % sucrose as described previously (Morrow et al. 2004b). The experiment was done in triplicate. a Flies survival after 48 h of indicated treatment. Data are expressed as mean ± SD. Statistical significance between each paraquat exposed groups and the control on sucrose was calculated using a one-way ANOVA followed by a Dunnett’s post hoc analysis (Prism). *p \ 0.05, and ****p \ 0.0001. b Whole fly protein extracts from a where separated on 12 % SDS polyacrylamide gel (SDS-PAGE) and transferred onto nitrocellulose membrane (Morrow et al. 2004b). Western blots were made with anti-Hsp22 (#36, 1/5000, (Morrow et al. 2000)), anti-Hsp60 (#37 1/5000, (Laplante et al. 1998)), anti-mtHsp70 (#JLD3, 1/5000, (Carbajal et al. 1993)), and a peroxidaseconjugated goat-anti-rabbit secondary antibody
regulated by ROS accumulation in the mitochondria and arguing for their participation in Drosophila UPRMT.
Drosophila UPRMT and Hsp22 are interconnected The UPRMT of Drosophila is not as well defined as the one in C. elegans described in the introduction. For instance, two mitochondrial proteases would be involved in flies UPRMT, namely CG4538 (the
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ortholog of ClpX) and CG5045 (the ortholog of ClpP) (Baqri et al. 2014; Owusu-Ansah et al. 2008). The way by which the signal is transduced from the mitochondria to the nucleus has not been investigated in details, but one study has shown an increased translocation of the transcription factor DVE (ortholog of C. elegans DVE-1) to the nucleus in conditions of mitochondrial stress, a situation similar to worms UPRMT (Baqri et al. 2014). However, another group has shown that part of the flies UPRMT is driven by the c-Jun Nterminal kinase (JNK) pathway through activation of jun-related antigen (Jra also known as Djun, the fly ortholog of c-Jun), which is similar to mammalian UPRMT (Hill and Van Remmen 2014; Owusu-Ansah et al. 2008). Interestingly, in flies the JNK pathway promotes lifespan through dFOXO (forkhead box, sub-group O) activation, and among the targets of this transcription factor there are many HSPs including Hsp22 (Demontis and Perrimon 2010; Harvey et al. 2008; Wang et al. 2003, 2005). Beside hsp22 and hsp60, hsp60C, and mthsp70 are the chaperones genes induced following mitochondrial stress in flies (Fernandez-Ayala et al. 2010; Owusu-Ansah et al. 2008). At the protein level, Hsp22 is robustly up-regulated upon mitochondrial stress induced by low dose of paraquat (Fig. 1). One peculiarity of this sHSP is that it can influence its own level of expression (Tower et al. 2014), and it was therefore suggested that Hsp22 could take part in the amplification of the UPRMT signal (Shen and Tower 2013; Tower 2014). The level of induction of Hsp60 expression depends on the cause of mitochondrial stress underlying the existence of more then one branch of UPRMT (Bennett and Kaeberlein 2014; Bennett et al. 2014; Hill and Van Remmen 2014; Jensen and Jasper 2014; Runkel et al. 2013, 2014). Indeed, while Hsp60 is mildly up-regulated by paraquat-induced UPRMT (Fig. 1b), it is greatly induced upon UPRMT triggered by over-expression of misfolding ornithine transcarbamylase in mitochondria (Pimenta de Castro et al. 2012). Of note, Drosophila HSP60 family comprises four members (Hsp60, Hsp60B, Hsp60C and Hsp60D) and the two members showing the greatest homology with human Hsp60 are also the ones that are required for lifespan extension by ETC perturbation and therefore involved in UPRMT (Owusu-Ansah et al. 2008). No change in mtHsp70 protein expression has yet been observed in Drosophila and mammals (Jensen and Jasper 2014;
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Owusu-Ansah et al. 2008). Accordingly, mtHsp70 is not induced in the same conditions as Hsp22 and Hsp60 upon mitochondrial treatment with paraquat suggesting different post-translational regulation of the chaperones and highlighting the need for further studies to characterize flies UPRMT (Fig. 1b). Alternatively, the late induction of the mtHsp70 signal may reflect the triggering of a more general cellular stress response since it corresponds to the timing of Hsp83 induction, which is cytoplasmic. Moreover, while the anti-mtHsp70 recognizes mainly the mitochondrial form of Hsp70, it recognizes also the nuclear form (Carbajal et al. 1993). Finally, the involvement of TRAP1 in the flies UPRMT has been suggested from studies using flies over-expressing the chaperone or carrying mutations preventing its expression but no direct measurements of its mRNA or protein levels upon UPRMT induction have yet been reported (Baqri et al. 2014).
UPRMT and Hsp22-mediated lifespan extension share important features One special feature of flies UPRMT is the triggering of a systemic activation of dFOXO through the secretion of insulin-like peptide binding protein ImpL2 (an ortholog of human IGFBP7), resulting in systemic inhibition of insulin/IGF signaling (IIS). Accordingly, UPRMT activation in muscles was shown to have repercussion in peripheral tissues and to promote healthy longevity (Owusu-Ansah et al. 2008). While the ImpL2 branch of mitochondrial stress defense would promote mitophagy, the concomitant dFOXO activation may also up-regulate the mitochondrial superoxide dismutase (MnSOD), which can in return induce UPRMT (Curtis et al. 2007; Demontis and Perrimon 2010; Honda and Honda 1999; Shen and Tower 2013; Tower 2014; Zarse et al. 2012). Interestingly, in addition to being a direct transcriptional target of dFOXO activity, Hsp22 is also induced by MnSOD over-expression (Curtis et al. 2007; Harvey et al. 2008; Tower et al. 2014). Furthermore, similarly to UPRMT activation in muscles, the targeted overexpression of Hsp22 in specific cell types was shown to have different effects on lifespan that were reflected at the organism level. Indeed, targeting Hsp22 overexpression in motor-neurons (with the D42 driver) was enough to increase flies health-span by 30 % as well as
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to confer resistance to robust oxidative stress (Morrow et al. 2004b). Moreover, flies genetically selected for their increased longevity, were shown to have increased levels of hsp22 RNA suggesting a role for this sHsp in aging determination (Kurapati et al. 2000). The relationship between UPRMT and lifespan is not always straightforward, and in C. elegans the UPRMT must be activated in the larval stage to have a beneficial effect on lifespan (Durieux et al. 2011; Houtkooper et al. 2013; Yoneda et al. 2004). This could partly be attributable to the burst of mitochondrial biogenesis at this stage (Tsang and Lemire 2002). In Drosophila, the time window for lifespan extension by UPRMT activation is less stringent and while RNAi of certain ETC components increase lifespan when performed in adults, other components only extend lifespan when removed through development from embryogenesis (Copeland et al. 2009; Owusu-Ansah et al. 2008). In the case of Hsp22, it was clearly shown that over-expression must be achieved before 4 days of age to get an increase in lifespan at physiological temperature (Bhole et al. 2004; Morrow et al. 2004b). Of note, the study of Bhole et al. has also shown that inducing Hsp22 expression with doxycycline at 4 days of age resulted in a greater sensitivity of flies to thermal and oxidative stress (Bhole et al. 2004). However, since doxycycline disturbs mitochondrial function, it is not clear to which extent Hsp22 by itself is responsible for the phenotype observed in this study and if the combined effects of doxycycline, stress and forced expression of a mitochondrial protein are also involved (Ballard and Melvin 2007; Moullan et al. 2015). Even if UPRMT activation does not always result in lifespan extension, it does improve flies fitness, which is also observed upon Hsp22 over-expression (Morrow et al. 2004b; OwusuAnsah et al. 2008). Evidence is emerging that changes to the production of other mitochondrial metabolic by-products brought by the UPRMT also have important physiological effects (Genova and Lenaz 2015; Jensen and Jasper 2014; Scialo et al. 2013). This is notably the case of ATP and NAD?, which can influence important cellular functions. Accordingly, it was suggested that UPRMT promotes longevity through its impact on NAD? metabolism (Jasper 2013; Mouchiroud et al. 2013). Among other enzymes, NAD? is the substrate of sirtuins deacetylases, which regulate energy
metabolism and have been shown to increase longevity upon over-expression (Guarente 2013; Jasper 2013; Mouchiroud et al. 2013). Interestingly, preliminary results from our laboratory suggest that there is less acetylated lysine in Hsp22 over-expressing flies and, in fact, there are important changes in the posttranslational modifications of mitochondrial proteins suggesting a different mitochondrial metabolism comparatively to normal lived flies (Morrow et al. submitted). Of note, Hsp22 over-expression also has influence on the transcriptional profile of flies (Kim et al. 2010).
Hsp22 expression prevents the accumulation of oxidatively-damaged proteins Hsp22 RNAi strains and fly lines carrying a p-element preventing normal Hsp22 expression are sensitive to oxidative stress induced by paraquat and H2O2 feeding, suggesting a protective function of the sHSP (Hirano et al. 2012; Morrow et al. 2004a; Moskalev et al. 2009). Indeed, in the case of hsp22 RNAi lines, it was shown that they develop memory defects prior to wild-type flies upon treatment with paraquat and H2O2 (Hirano et al. 2012). Additionally, in the case of lines bearing a p-element in the promoter of hsp22, it was shown that their survival to paraquat feeding was greatly diminished (Morrow et al. 2004a; Moskalev et al. 2009). Accordingly, flies over-expressing Hsp22 with the Gal4/UAS system are more resistant to oxidative stress induced by paraquat (Morrow et al. 2004b). The exact mechanism by which Hsp22 exert its beneficial effects is not well defined. The simplest explanation is that it insures proteostasis through its chaperone function. Results from studies using primary human fibroblasts transfected with a retrovirus encoding Drosophila Hsp22, suggest that its clients are conserved in orthologous systems (Wadhwa et al. 2010). Indeed, over-expression of Hsp22 in those cells resulted in an increase in population doublings from 58 to 84 accompanied by lower levels of the senescence associated ß-galactosidase marker. It was also shown that p53 co-immunoprecipitates with Hsp22 in these cells and accordingly, p53 was found in mitochondria of Hsp22 over-expressing cells (Wadhwa et al. 2010).
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A
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Fig. 2 Hsp22 over-expression does not affect the assembly of ETC complexes. Crude mitochondria were isolated from 1 dayold flies over-expressing Hsp22 ubiquitously with the Gal4/ UAS system (Hsp22?) and corresponding controls (c) using successive centrifugations at 1000 and 8800 g [Morrow et al. submitted, (Melov et al. 1999; Morrow et al. 2004b)]. The UASHsp22 fly strain has been described previously (Morrow et al. 2004b; Rorth 1996) and carries a UAS containing P-element approximately 640 bp upstream hsp22 ATG. Mitochondrial membranes proteins were solubilized by resuspending crude mitochondria in digitonin buffer (12.5 mM bis-Tris, 1.5 N HCl,
12.5 mM NaCl, 1 % digitonin). Equal amounts of 20,000 g supernatants were then applied on blue native-PAGE according to manufacturer instructions (Life Technologies) (a) and the lanes were taken for a successive SDS-PAGE dimension (b) (Krause 2006; Krause and Seelert 2008). a In the first dimension, individual proteins, protein complexes and supercomplexes are separated in their native state. b In the second dimension, proteins are denatured and complexes disassembled allowing subunits of a given complex to migrate as a vertical line. Control (C): actin5C-Gal4 flies, Hsp22?: actin5CGal4;EP(3)3247 flies (Hsp22 over-expressing flies)
Interestingly, flies over-expressing Hsp22 have higher mitochondrial protease activity relatively to control flies at the beginning of adulthood and lower levels of carbonylated-proteins suggesting a better turnover of oxidatively-damaged proteins (Morrow et al. submitted). Moreover, it was shown that cells expressing an hsp22-GFP reporter construct have low level of mitochondrial ROS (Tower et al. 2014). While this has been proposed to be the result of the repression of mitochondrial metabolic activity by Hsp22 expression, another possibility could be that Hsp22 can chaperone the individual respiratory complexes to form stable assemblages thus preventing electron leakage and subsequent ROS production in a way similar to yeast Aac2 and mammalian Rcf1 and Rcf2 (Tower et al. 2014; Winge 2012). In such a scenario, it is important to mention that Hsp22 would act to improve an existing assembly rather then being in the assembly per se. This is consistent with the observation that there is no change in the ETC complex profiles between control and Hsp22 over-expressing
flies (Fig. 2). Interestingly, we have observed an interaction of Hsp22 with subunit VIIa of complex IV, which is involved in complex III and IV interaction, as well as with CG4169 (ortholog of human UQCRC2) and ATPsyn-b (ortholog of human ATP5F1), which are respectively from complex III and V on the matrix side juxtaposed to the membranes (Morrow et al. in preparation, (Winge 2012)). Alternatively, Hsp22 may decrease ROS accumulation in the mitochondria by chaperoning complex I like plant mitochondrial sHSPs (Downs and Heckathorn 1998). Such interactions of Hsp22 with subunits from the ETC complexes would be in line with the presence of a small fraction of Hsp22 with mitochondrial membrane proteins upon mitochondrial fractionation (Morrow et al. 2000).
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Concluding remarks The relationship between ROS and aging is still not well understood. Recent studies suggest that cytoplasmic
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ROS are toxic while mitochondrial ROS mainly produced by complex I determines longevity (Barja 2014; Genova and Lenaz 2015; Ristow and Schmeisser 2011, 2014; Schaar et al. 2015; Scialo et al. 2013). In C. elegans, mitochondrial ROS and UPRMT have been shown to affect lifespan differently since their effect on lifespan is fully additive (Yang and Hekimi 2010). While UPRMT involves mitochondrial peptides exportation and ATFS-1 activation (Aldridge et al. 2007; Cristina et al. 2009; Haynes et al. 2007; Haynes and Ron 2010; Haynes et al. 2010; Jovaisaite et al. 2014; Nargund et al. 2012), mitochondrial ROS would relay their signal through proteins involved in the intrinsic apoptotic pathway independently of apoptosis (Yee et al. 2014). There is now evidence that there is more than one branch of UPRMT, which do not all results in increase longevity upon activation (Baker et al. 2012; Bennett and Kaeberlein 2014; Bennett et al. 2014; Nargund et al. 2012; Runkel et al. 2013, 2014). In fact some aspects of the UPRMT are limited by the nature of the stress, the age and the sex of the animal (Tower 2014). Accordingly, flies UPRMT driven by TRAP1 is different in males and females due partly to differences in bioenergetics between both sexes (Ballard et al. 2007; Baqri et al. 2014). Multiple evidence link Hsp22 to the UPRMT and concomitantly to the aging process. One goal of the UPRMT is to allow mitochondria to recover from the accumulation of damaged proteins in order to reestablish proteostasis. Hsp22 is a good candidate to accomplish this task due to its localization and to its effect on mitochondrial ROS accumulation. The association of Hsp22 with subunits from ETC complexes is exciting and worth to be examined further. The same is true for the interaction of Hsp22 with p53 since mitochondrial p53 can trigger apoptosis or inhibit autophagy depending on the nature of the stress (Green and Kroemer 2009; Levine and Oren 2009). Other partners of Hsp22 obtained in projects such as the Drosophila Protein Interaction Map will need further investigations [DPiM, (Guruharsha et al. 2011)]. Interestingly, many of these are involved in translation, proteolysis and autophagy and therefore stay in the same theme of promoting mitochondrial homeostasis. In summary, the beneficial effects of Hsp22 overexpression on lifespan and resistance to stress and its
involvement in flies UPRMT point to the important role of this sHSP on mitochondrial function. Acknowledgments This work was supported by a grant (MOP-111268) from the Canadian Institutes of Health Research to RMT. Complaince with Ethical Standards Conflict of interest The authors declare that they have no conflict of interest.
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REVIEW ARTICLE
Drosophila melanogaster mitochondrial Hsp22: a role in resistance to oxidative stress, aging and the mitochondrial unfolding protein response Genevie`ve Morrow . Marie Le Pe´cheur . Robert M. Tanguay
Received: 13 March 2015 / Accepted: 1 July 2015 Ó Springer Science+Business Media Dordrecht 2015
Abstract Aging is characterized by the accumulation of dysfunctional mitochondria. Since these organelles are involved in many important cellular processes, different mechanisms exist to maintain their integrity. Among them is the mitochondrial unfolding protein response, which triggers the expression of a set of proteins aimed at re-establishing mitochondrial homeostasis. The induction of mitochondrial chaperones expression, particularly of Hsp60 and Hsp70, is a hallmark of this pathway. In Drosophila melanogaster, Hsp22 is also up-regulated by mitochondrial stress. This small heat shock protein is one of the members of the family to be localized inside mitochondria. One characteristic of Drosophila Hsp22 is its preferential up-regulation during aging and in oxidative stress conditions. It is a beneficial protein since its over-expression increases lifespan and resistance to stress while its down-regulation is detrimental. This review focuses on Drosophila Hsp22 and its links with the mitochondrial unfolding protein response and the aging process, in addition to highlight the important role of this sHSP in mitochondrial homeostasis. G. Morrow M. Le Pe´cheur R. M. Tanguay (&) Laboratoire de Ge´ne´tique Cellulaire et De´veloppementale, De´partement de biologie mole´culaire, biochimie me´dicale et pathologie, Institute de Biologie Inte´grative et des Syste`mes (IBIS) and PROTEO, Universite´ Laval, Pavillon CE-Marchand, 1030 avenue de la me´decine, Que´bec, QC G1V 0A6, Canada e-mail: [email protected]
Keywords Hsp22 Small heat shock protein (sHSP) Oxidative stress Reactive oxygen species (ROS) Mitochondrial unfolding protein response (UPRMT) Aging
Introduction Aging is characterized by a decline of cellular functions that ultimately leads to death. The molecular mechanisms underlying this process are not all understood and multiple theories have been proposed to explain it [reviewed in (Vina et al. 2007; Zhou et al. 2011)]. Among them the mitochondrial free radical theory of aging has been intensively studied in the past decades and places mitochondria at the center of the aging process. According to this theory, reactive oxygen species (ROS) produced as by-products of mitochondrial ATP synthesis are causing damages to DNA, lipids and proteins that eventually lead to cellular dysfunction and death. This theory has been questioned recently due mainly to the failure of antioxidant therapies to extend aging and to the existence of organisms like the naked mole rat which have low levels of antioxidants and high levels of oxidative damage but nevertheless live seven times longer than the domestic mouse [reviewed in (Scialo et al. 2013)]. At physiological levels, it is now admitted that ROS signal information through oxidation of specific sulfhydryl groups on key signaling proteins (Genova
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and Lenaz 2015; Orr et al. 2013; Wang et al. 2012). However there is a threshold above which ROS become harmful and induce oxidative stress (Dai et al. 2014; Genova and Lenaz 2015; Orr et al. 2013; Rigoulet et al. 2011). Mitochondria are the main generators of ROS, and complex I is considered as the major source of ROS involved in aging (Barja 2014; Genova and Lenaz 2015; Holzerova and Prokisch 2015; Orr et al. 2013; Scialo et al. 2013). Indeed, longlived animals have species-specific low mitochondrial ROS generation rates at complex I, and the extension of longevity by caloric restriction can be mimicked by inhibition of this ETC complex [reviewed in (Barja 2014; Genova and Lenaz 2015; Ristow and Schmeisser 2011, 2014; Scialo et al. 2013)]. Interestingly, a recent study in Caenorhabditis elegans has shown that mitochondrial ROS can increase longevity in presence of a detrimental level of cytoplasmic ROS, demonstrating that ROS have a compartment specific effect on lifespan (Schaar et al. 2015). Mitochondria are essential organelles that are involved in multiple cellular processes. In addition to producing ATP through oxidative phosphorylation, they are central executioner of apoptosis and involved in calcium homeostasis/storage as well as in multiple anabolic and catabolic processes such as fatty acid ßoxidation, branch-chain amino-acid metabolism, heme and hormone synthesis and ammonia detoxification. Therefore, mitochondria have to adapt to the cell needs and are in constant communication with the nucleus (Haynes et al. 2007; Haynes and Ron 2010; Runkel et al. 2014; Schieke and Finkel 2006). While dysfunctions of mitochondria have been reported in numerous diseases as well as in aging, up-regulation of the mitochondrial stress response has gained recognition as a potential pro-longevity mechanism [reviewed in (Dai et al. 2014; Hill and Van Remmen 2014; Ziegler et al. 2015)]. This specific stress response includes changes in mitochondrial dynamic (fission/fusion), regulation of mitochondrial biogenesis/turnover, retrograde signaling and mitochondrial unfolding protein response (UPRMT) [reviewed in (Hamon et al. 2015; Hill and Van Remmen 2014; Ikeda et al. 2015; Jensen and Jasper 2014; Vilchez et al. 2014)]. The UPRMT is a mitochondrial-to-nuclear signal transduction pathway initiated by the accumulation of unfolded proteins in mitochondria and resulting in the induction of stress response proteins such as anti-
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oxidants, chaperones and proteases. It has first been observed in C. elegans but is now recognized in other eukaryotes although it is not as well defined (Haynes et al. 2007; Haynes and Ron 2010; Hill and Van Remmen 2014; Jensen and Jasper 2014). In C. elegans, mitochondrial unfolded proteins are cleaved by caseinolytic protease P (ClpP), an ATPdependent mitochondrial matrix protease. The resulting peptides are then exported out of the mitochondria to the cytosol by HAF-1 (an ATP binding cassette transporter) where they weaken mitochondrial import, thereby preventing ATFS-1 (activating transcription factor associated with stress 1) transcription factor to be imported into the mitochondria and degraded by the Lon protease. Consequently, ATFS-1 translocates to the nucleus and activates mitochondrial stress response genes in cooperation with ubiquitin-like 5 (UBL-5) and DVE-1 (an homeodomain-containing transcription factor) to ultimately reconstitute mitochondrial homeostasis (Aldridge et al. 2007; Cristina et al. 2009; Haynes et al. 2007; Haynes and Ron 2010; Haynes et al. 2010; Jovaisaite et al. 2014; Nargund et al. 2012). Among mitochondrial chaperones, Hsp60/Hsp10 and mitochondrial Hsp70 (mtHsp70) have been associated with UPRMT in different organisms. In Drosophila melanogaster, two other mitochondrial chaperones are involved in this important stress response, namely Hsp22 and tumor necrosis factor receptor associated protein 1 (TRAP1) (Baqri et al. 2014; Shen and Tower 2013; Tower 2014; Tower et al. 2014). While TRAP1 is a member of the HSP90 family of chaperone, Hsp22 is a small heat shock protein (sHSP) that is preferentially up-regulated during aging. We will here review the evidence linking D. melanogaster Hsp22 to flies UPRMT and aging processes.
Hsp22 is a mitochondrial chaperone induced by mitochondrial stress In D. melanogaster there are 12 sHSPs, which have distinctive developmental expression pattern and intracellular localization [reviewed in (Morrow and Tanguay 2015)]. Among them, Hsp22 is the only one to be found in mitochondria. More precisely it is located in the mitochondrial matrix although a small fraction has been shown to sediment with
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A Survival following 48 hours of paraquat exposure (%)
mitochondrial membrane proteins especially after stress (Morrow et al. 2000). As most members of the sHSP family, Hsp22 prevents in vitro heat-induced protein aggregation and maintains heat-denatured protein in a refoldable state for the ATP-dependent chaperones such as Hsp60 and Hsp70 (Morrow et al. 2006). In vivo, its chaperone activity has not been assayed directly, but as other sHSPs it has been shown to interact with proteins involved in a wide range of cellular functions suggesting that its chaperone function is not restricted to specific clients (Basha et al. 2004; Giot et al. 2003; Guruharsha et al. 2011; Haslbeck and Vierling 2015). During development, Hsp22 is expressed in the course of metamorphosis from larvae to pupae and is up-regulated during aging (King and Tower 1999; Michaud et al. 2002). Importantly, Hsp22 is also robustly up-regulated upon mitochondrial stress. Indeed, hsp22 is one of the most up-regulated genes in tko25 flies that have an insufficient mitochondrial translational capacity due to a mutation in mitoribosomal protein S12 (mRpS12) (Fernandez-Ayala et al. 2010). Moreover, hsp22 together with l(2)efl (another sHSP from Drosophila), are the sHSPs genes showing the greatest induction upon oxidative stress (H2O2, paraquat, C99.5 % O2 atmosphere (Girardot et al. 2006; Gruenewald et al. 2009; Hirano et al. 2012; Landis et al. 2004, 2012). Reagents that induce ROS production are recognized activator of UPRMT, together with interventions that deplete the mitochondrial genome, delete mitochondrial electron transport chain (ETC) genes and inhibit mitochondrial protease activity (Aldridge et al. 2007; Copeland et al. 2009; Durieux et al. 2011; Haynes and Ron 2010; Jensen and Jasper 2014; Yoneda et al. 2004). Paraquat is a methyl viologen that generates superoxide through ETC complex I inhibition. While it is detrimental at high dose it has been shown to increase lifespan at low dose, highlighting the potential of ROS as signaling molecules [reviewed in (Ristow and Schmeisser 2011, 2014)]. Interestingly, hsp22 expression is induced following both robust and mild oxidative stresses brought by paraquat (Hirano et al. 2012). At the protein level, Hsp22 is up-regulated in presence of 1 mM paraquat, a dose which do not significantly alter fly survival (Fig. 1). Of note is the concomitant but very mild induction of Hsp60 in the same conditions suggesting that both heat shock proteins (HSPs) can be up-
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Fig. 1 Differential induction of mitochondrial chaperones upon oxidative stress mediated by paraquat. Five days-old W1118 flies (60–80/condition) were starved (no food) for 6 h and then put in presence of a piece of Whatman 3 M filter soaked with 200 ll of 1 % sucrose (control, 0) or different concentrations of paraquat diluted in 1 % sucrose as described previously (Morrow et al. 2004b). The experiment was done in triplicate. a Flies survival after 48 h of indicated treatment. Data are expressed as mean ± SD. Statistical significance between each paraquat exposed groups and the control on sucrose was calculated using a one-way ANOVA followed by a Dunnett’s post hoc analysis (Prism). *p \ 0.05, and ****p \ 0.0001. b Whole fly protein extracts from a where separated on 12 % SDS polyacrylamide gel (SDS-PAGE) and transferred onto nitrocellulose membrane (Morrow et al. 2004b). Western blots were made with anti-Hsp22 (#36, 1/5000, (Morrow et al. 2000)), anti-Hsp60 (#37 1/5000, (Laplante et al. 1998)), anti-mtHsp70 (#JLD3, 1/5000, (Carbajal et al. 1993)), and a peroxidaseconjugated goat-anti-rabbit secondary antibody
regulated by ROS accumulation in the mitochondria and arguing for their participation in Drosophila UPRMT.
Drosophila UPRMT and Hsp22 are interconnected The UPRMT of Drosophila is not as well defined as the one in C. elegans described in the introduction. For instance, two mitochondrial proteases would be involved in flies UPRMT, namely CG4538 (the
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ortholog of ClpX) and CG5045 (the ortholog of ClpP) (Baqri et al. 2014; Owusu-Ansah et al. 2008). The way by which the signal is transduced from the mitochondria to the nucleus has not been investigated in details, but one study has shown an increased translocation of the transcription factor DVE (ortholog of C. elegans DVE-1) to the nucleus in conditions of mitochondrial stress, a situation similar to worms UPRMT (Baqri et al. 2014). However, another group has shown that part of the flies UPRMT is driven by the c-Jun Nterminal kinase (JNK) pathway through activation of jun-related antigen (Jra also known as Djun, the fly ortholog of c-Jun), which is similar to mammalian UPRMT (Hill and Van Remmen 2014; Owusu-Ansah et al. 2008). Interestingly, in flies the JNK pathway promotes lifespan through dFOXO (forkhead box, sub-group O) activation, and among the targets of this transcription factor there are many HSPs including Hsp22 (Demontis and Perrimon 2010; Harvey et al. 2008; Wang et al. 2003, 2005). Beside hsp22 and hsp60, hsp60C, and mthsp70 are the chaperones genes induced following mitochondrial stress in flies (Fernandez-Ayala et al. 2010; Owusu-Ansah et al. 2008). At the protein level, Hsp22 is robustly up-regulated upon mitochondrial stress induced by low dose of paraquat (Fig. 1). One peculiarity of this sHSP is that it can influence its own level of expression (Tower et al. 2014), and it was therefore suggested that Hsp22 could take part in the amplification of the UPRMT signal (Shen and Tower 2013; Tower 2014). The level of induction of Hsp60 expression depends on the cause of mitochondrial stress underlying the existence of more then one branch of UPRMT (Bennett and Kaeberlein 2014; Bennett et al. 2014; Hill and Van Remmen 2014; Jensen and Jasper 2014; Runkel et al. 2013, 2014). Indeed, while Hsp60 is mildly up-regulated by paraquat-induced UPRMT (Fig. 1b), it is greatly induced upon UPRMT triggered by over-expression of misfolding ornithine transcarbamylase in mitochondria (Pimenta de Castro et al. 2012). Of note, Drosophila HSP60 family comprises four members (Hsp60, Hsp60B, Hsp60C and Hsp60D) and the two members showing the greatest homology with human Hsp60 are also the ones that are required for lifespan extension by ETC perturbation and therefore involved in UPRMT (Owusu-Ansah et al. 2008). No change in mtHsp70 protein expression has yet been observed in Drosophila and mammals (Jensen and Jasper 2014;
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Owusu-Ansah et al. 2008). Accordingly, mtHsp70 is not induced in the same conditions as Hsp22 and Hsp60 upon mitochondrial treatment with paraquat suggesting different post-translational regulation of the chaperones and highlighting the need for further studies to characterize flies UPRMT (Fig. 1b). Alternatively, the late induction of the mtHsp70 signal may reflect the triggering of a more general cellular stress response since it corresponds to the timing of Hsp83 induction, which is cytoplasmic. Moreover, while the anti-mtHsp70 recognizes mainly the mitochondrial form of Hsp70, it recognizes also the nuclear form (Carbajal et al. 1993). Finally, the involvement of TRAP1 in the flies UPRMT has been suggested from studies using flies over-expressing the chaperone or carrying mutations preventing its expression but no direct measurements of its mRNA or protein levels upon UPRMT induction have yet been reported (Baqri et al. 2014).
UPRMT and Hsp22-mediated lifespan extension share important features One special feature of flies UPRMT is the triggering of a systemic activation of dFOXO through the secretion of insulin-like peptide binding protein ImpL2 (an ortholog of human IGFBP7), resulting in systemic inhibition of insulin/IGF signaling (IIS). Accordingly, UPRMT activation in muscles was shown to have repercussion in peripheral tissues and to promote healthy longevity (Owusu-Ansah et al. 2008). While the ImpL2 branch of mitochondrial stress defense would promote mitophagy, the concomitant dFOXO activation may also up-regulate the mitochondrial superoxide dismutase (MnSOD), which can in return induce UPRMT (Curtis et al. 2007; Demontis and Perrimon 2010; Honda and Honda 1999; Shen and Tower 2013; Tower 2014; Zarse et al. 2012). Interestingly, in addition to being a direct transcriptional target of dFOXO activity, Hsp22 is also induced by MnSOD over-expression (Curtis et al. 2007; Harvey et al. 2008; Tower et al. 2014). Furthermore, similarly to UPRMT activation in muscles, the targeted overexpression of Hsp22 in specific cell types was shown to have different effects on lifespan that were reflected at the organism level. Indeed, targeting Hsp22 overexpression in motor-neurons (with the D42 driver) was enough to increase flies health-span by 30 % as well as
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to confer resistance to robust oxidative stress (Morrow et al. 2004b). Moreover, flies genetically selected for their increased longevity, were shown to have increased levels of hsp22 RNA suggesting a role for this sHsp in aging determination (Kurapati et al. 2000). The relationship between UPRMT and lifespan is not always straightforward, and in C. elegans the UPRMT must be activated in the larval stage to have a beneficial effect on lifespan (Durieux et al. 2011; Houtkooper et al. 2013; Yoneda et al. 2004). This could partly be attributable to the burst of mitochondrial biogenesis at this stage (Tsang and Lemire 2002). In Drosophila, the time window for lifespan extension by UPRMT activation is less stringent and while RNAi of certain ETC components increase lifespan when performed in adults, other components only extend lifespan when removed through development from embryogenesis (Copeland et al. 2009; Owusu-Ansah et al. 2008). In the case of Hsp22, it was clearly shown that over-expression must be achieved before 4 days of age to get an increase in lifespan at physiological temperature (Bhole et al. 2004; Morrow et al. 2004b). Of note, the study of Bhole et al. has also shown that inducing Hsp22 expression with doxycycline at 4 days of age resulted in a greater sensitivity of flies to thermal and oxidative stress (Bhole et al. 2004). However, since doxycycline disturbs mitochondrial function, it is not clear to which extent Hsp22 by itself is responsible for the phenotype observed in this study and if the combined effects of doxycycline, stress and forced expression of a mitochondrial protein are also involved (Ballard and Melvin 2007; Moullan et al. 2015). Even if UPRMT activation does not always result in lifespan extension, it does improve flies fitness, which is also observed upon Hsp22 over-expression (Morrow et al. 2004b; OwusuAnsah et al. 2008). Evidence is emerging that changes to the production of other mitochondrial metabolic by-products brought by the UPRMT also have important physiological effects (Genova and Lenaz 2015; Jensen and Jasper 2014; Scialo et al. 2013). This is notably the case of ATP and NAD?, which can influence important cellular functions. Accordingly, it was suggested that UPRMT promotes longevity through its impact on NAD? metabolism (Jasper 2013; Mouchiroud et al. 2013). Among other enzymes, NAD? is the substrate of sirtuins deacetylases, which regulate energy
metabolism and have been shown to increase longevity upon over-expression (Guarente 2013; Jasper 2013; Mouchiroud et al. 2013). Interestingly, preliminary results from our laboratory suggest that there is less acetylated lysine in Hsp22 over-expressing flies and, in fact, there are important changes in the posttranslational modifications of mitochondrial proteins suggesting a different mitochondrial metabolism comparatively to normal lived flies (Morrow et al. submitted). Of note, Hsp22 over-expression also has influence on the transcriptional profile of flies (Kim et al. 2010).
Hsp22 expression prevents the accumulation of oxidatively-damaged proteins Hsp22 RNAi strains and fly lines carrying a p-element preventing normal Hsp22 expression are sensitive to oxidative stress induced by paraquat and H2O2 feeding, suggesting a protective function of the sHSP (Hirano et al. 2012; Morrow et al. 2004a; Moskalev et al. 2009). Indeed, in the case of hsp22 RNAi lines, it was shown that they develop memory defects prior to wild-type flies upon treatment with paraquat and H2O2 (Hirano et al. 2012). Additionally, in the case of lines bearing a p-element in the promoter of hsp22, it was shown that their survival to paraquat feeding was greatly diminished (Morrow et al. 2004a; Moskalev et al. 2009). Accordingly, flies over-expressing Hsp22 with the Gal4/UAS system are more resistant to oxidative stress induced by paraquat (Morrow et al. 2004b). The exact mechanism by which Hsp22 exert its beneficial effects is not well defined. The simplest explanation is that it insures proteostasis through its chaperone function. Results from studies using primary human fibroblasts transfected with a retrovirus encoding Drosophila Hsp22, suggest that its clients are conserved in orthologous systems (Wadhwa et al. 2010). Indeed, over-expression of Hsp22 in those cells resulted in an increase in population doublings from 58 to 84 accompanied by lower levels of the senescence associated ß-galactosidase marker. It was also shown that p53 co-immunoprecipitates with Hsp22 in these cells and accordingly, p53 was found in mitochondria of Hsp22 over-expressing cells (Wadhwa et al. 2010).
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A
BN-PAGE
B
I1III2 V2 I1 V1III2 IV1
SDS-PAGE (kDa)
I1III2IV1-3 I1III2 V2 I1 V1
III2
I1III2 V2 I1 V1III2 IV1
97 66 45 30
IV1 20 14.4
C Hsp 22+
Control
Hsp22+
Fig. 2 Hsp22 over-expression does not affect the assembly of ETC complexes. Crude mitochondria were isolated from 1 dayold flies over-expressing Hsp22 ubiquitously with the Gal4/ UAS system (Hsp22?) and corresponding controls (c) using successive centrifugations at 1000 and 8800 g [Morrow et al. submitted, (Melov et al. 1999; Morrow et al. 2004b)]. The UASHsp22 fly strain has been described previously (Morrow et al. 2004b; Rorth 1996) and carries a UAS containing P-element approximately 640 bp upstream hsp22 ATG. Mitochondrial membranes proteins were solubilized by resuspending crude mitochondria in digitonin buffer (12.5 mM bis-Tris, 1.5 N HCl,
12.5 mM NaCl, 1 % digitonin). Equal amounts of 20,000 g supernatants were then applied on blue native-PAGE according to manufacturer instructions (Life Technologies) (a) and the lanes were taken for a successive SDS-PAGE dimension (b) (Krause 2006; Krause and Seelert 2008). a In the first dimension, individual proteins, protein complexes and supercomplexes are separated in their native state. b In the second dimension, proteins are denatured and complexes disassembled allowing subunits of a given complex to migrate as a vertical line. Control (C): actin5C-Gal4 flies, Hsp22?: actin5CGal4;EP(3)3247 flies (Hsp22 over-expressing flies)
Interestingly, flies over-expressing Hsp22 have higher mitochondrial protease activity relatively to control flies at the beginning of adulthood and lower levels of carbonylated-proteins suggesting a better turnover of oxidatively-damaged proteins (Morrow et al. submitted). Moreover, it was shown that cells expressing an hsp22-GFP reporter construct have low level of mitochondrial ROS (Tower et al. 2014). While this has been proposed to be the result of the repression of mitochondrial metabolic activity by Hsp22 expression, another possibility could be that Hsp22 can chaperone the individual respiratory complexes to form stable assemblages thus preventing electron leakage and subsequent ROS production in a way similar to yeast Aac2 and mammalian Rcf1 and Rcf2 (Tower et al. 2014; Winge 2012). In such a scenario, it is important to mention that Hsp22 would act to improve an existing assembly rather then being in the assembly per se. This is consistent with the observation that there is no change in the ETC complex profiles between control and Hsp22 over-expressing
flies (Fig. 2). Interestingly, we have observed an interaction of Hsp22 with subunit VIIa of complex IV, which is involved in complex III and IV interaction, as well as with CG4169 (ortholog of human UQCRC2) and ATPsyn-b (ortholog of human ATP5F1), which are respectively from complex III and V on the matrix side juxtaposed to the membranes (Morrow et al. in preparation, (Winge 2012)). Alternatively, Hsp22 may decrease ROS accumulation in the mitochondria by chaperoning complex I like plant mitochondrial sHSPs (Downs and Heckathorn 1998). Such interactions of Hsp22 with subunits from the ETC complexes would be in line with the presence of a small fraction of Hsp22 with mitochondrial membrane proteins upon mitochondrial fractionation (Morrow et al. 2000).
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Concluding remarks The relationship between ROS and aging is still not well understood. Recent studies suggest that cytoplasmic
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ROS are toxic while mitochondrial ROS mainly produced by complex I determines longevity (Barja 2014; Genova and Lenaz 2015; Ristow and Schmeisser 2011, 2014; Schaar et al. 2015; Scialo et al. 2013). In C. elegans, mitochondrial ROS and UPRMT have been shown to affect lifespan differently since their effect on lifespan is fully additive (Yang and Hekimi 2010). While UPRMT involves mitochondrial peptides exportation and ATFS-1 activation (Aldridge et al. 2007; Cristina et al. 2009; Haynes et al. 2007; Haynes and Ron 2010; Haynes et al. 2010; Jovaisaite et al. 2014; Nargund et al. 2012), mitochondrial ROS would relay their signal through proteins involved in the intrinsic apoptotic pathway independently of apoptosis (Yee et al. 2014). There is now evidence that there is more than one branch of UPRMT, which do not all results in increase longevity upon activation (Baker et al. 2012; Bennett and Kaeberlein 2014; Bennett et al. 2014; Nargund et al. 2012; Runkel et al. 2013, 2014). In fact some aspects of the UPRMT are limited by the nature of the stress, the age and the sex of the animal (Tower 2014). Accordingly, flies UPRMT driven by TRAP1 is different in males and females due partly to differences in bioenergetics between both sexes (Ballard et al. 2007; Baqri et al. 2014). Multiple evidence link Hsp22 to the UPRMT and concomitantly to the aging process. One goal of the UPRMT is to allow mitochondria to recover from the accumulation of damaged proteins in order to reestablish proteostasis. Hsp22 is a good candidate to accomplish this task due to its localization and to its effect on mitochondrial ROS accumulation. The association of Hsp22 with subunits from ETC complexes is exciting and worth to be examined further. The same is true for the interaction of Hsp22 with p53 since mitochondrial p53 can trigger apoptosis or inhibit autophagy depending on the nature of the stress (Green and Kroemer 2009; Levine and Oren 2009). Other partners of Hsp22 obtained in projects such as the Drosophila Protein Interaction Map will need further investigations [DPiM, (Guruharsha et al. 2011)]. Interestingly, many of these are involved in translation, proteolysis and autophagy and therefore stay in the same theme of promoting mitochondrial homeostasis. In summary, the beneficial effects of Hsp22 overexpression on lifespan and resistance to stress and its
involvement in flies UPRMT point to the important role of this sHSP on mitochondrial function. Acknowledgments This work was supported by a grant (MOP-111268) from the Canadian Institutes of Health Research to RMT. Complaince with Ethical Standards Conflict of interest The authors declare that they have no conflict of interest.
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