MINIREVIEW
Dual localization of glutathione S -transferase in the
cytosol and mitochondria:implications in oxidative stress,toxicity and disease
Haider Raza
Department of Biochemistry,Faculty of Medicine and Health Sciences,UAE University,Al Ain,UAE
Keywords
chemical toxicity;cryptic mitochondrial
signal;dual localization of GSTs;glutathione S -transferase;Hsp70chaperone;N-terminal signal region;oxidative stress;PKA phosphorylation
Correspondence
Haider Raza,Department of Biochemistry,Faculty of Medicine and Health Sciences,UAE University,PO Box 17666,Al Ain,UAE.
Fax:+97137672033Tel:+97137137506
E-mail:h.raza@uaeu.ac.ae
(Received 19May 2011,revised 12August 2011,accepted 12September 2011)doi:10.1111/j.1742-4658.2
011.08358.x
Glutathione (GSH)conjugating enzymes,glutathione S -transferases (GSTs),are present in different subcellular compartments including cytosol,mitochondria,endoplasmic reticulum,nucleus and plasma membrane.The regulation and function of GSTs have implications in cell growth,oxidative stress as well as disease progression and prevention.Of the several mitochondria localized forms,GSTK (GST kappa)is mitochondria-specific since it contains N-terminal canonical and cleavable mitochondria targeting signals.Other forms like GST alpha,mu and pi purified from mitochondria are similar to the cytosolic molecular forms or ‘echoproteins’.Altered GST expression has been implicated in hepatic,cardiac and neurological diseases.Mitochondria-specific GSTK has also been implicated in obesity,diabetes and related metabolic disorders.Studies have shown that silencing the GSTA4(GST alpha)gene resulted in mitochondrial dysfunction,as was also seen in GSTA4null mice,which could contribute to insulin resistance in type 2diabetes.This review highlights the significance of the mitochon-drial GST pool,particularly the mechanism and significance of dual target-ing of GSTA4-4under in vitro and in vivo conditions.GSTA4-4is targeted in the mitochondria by activation of the internal cryptic signal present at the C-terminus of the protein by protein-kinase-dependent phosphorylation and cytosolic heat shock protein (Hsp70)chaperone.Mitochondrial GST pi,on the other
hand,has been shown to have two uncleaved cryptic signals rich in positively charged amino acids at the N-terminal region.Both physiological and pathophysiological implications of GST translocation to mitochondria are discussed in the review.
Introduction
The induced expression of multiple forms of glutathi-one S -transferase (GST;EC 2.5.1.18)appears to be an evolutionary response of cells for protection against chemical toxicity and oxidative stress.The tissue-and species-specific expression and distribution of GSTs
are considered to be an adaptive response against the toxicity of endogenous and exogenous metabolites [1,2]and oxidative stress related degenerative disorders,and in drug resistance seen in cancer therapy.GST iso-enzymes have also been implicated in the progression
Abbreviations
cGSH,cytosolic GSH;cGST,cytosolic GST;CYP,cytochrome P450;ER,endoplasmic reticulum;GSH,glutathione;GSSG,oxidized glutathione;GST,glutathione S -transferase;4-HNE,4-hydroxynonenal;Hsp,heat shock proteins;MAP,mitogen-activated protein;mGSH,mitochondrial GSH;mGST,mitochondrial GST;ROS,reactive oxygen species;UCP,uncoupling protein.
of cancer[1–4].GSTs play an important role in the activation of signals by mitogen-activated protein (MAP)kinases and various transcription factors that regulate apoptosis and cell survival[4,5].Maintenance of the cellular antioxidant glutathione(GSH)in differ-ent cellular compartments is also critically regulated by GSTs.GST is a multifunctional enzyme involved in cellular detoxification of endogenous toxic metabolites, superoxide radicals and exogenous toxic chemicals [3,4].The ubiquitous distribution of GSTs in microbes, animals and plants signifies the physiological impor-tance of this multigene family of enzymes.
GSTs are divided into soluble cytosolic[cGST alpha (A,a),mu(M,l),Pi(pi,p),omega(x),theta(T,h), delta(d),sigma(r),zeta(f)],mitochondrial[mGST a, l,p and kappa(K)]and structurally distinct mem-brane-bound microsomal GST(MGST).The MGST family contains six members including1,2,3,leukotri-ene C-4synthase,5-lipoxygenase activating protein and prostaglandin E synthase,now referred to as membrane-associated proteins in eicosanoid and gluta-thione metabolism members[1,2].Each member of the family has multiple isoenzymes with overlapping sub-strate specificity.GSTs with some specific catalytic properties have also been reported in the plasma mem-brane,outer mitochondrial membrane as well as in the nucleus[1–3].Several groups,including ours,have suc-cessfully purified and characterized multiple forms of GSTs from hepatic mitochondria[6–10].Recently, dual localization o
f GSTK in the peroxisome and mitochondria has been reported[10].These mitochon-drial GST(mGST)proteins are coded by nuclear genes,synthesized in the cytoplasm and then trans-ported to mitochondria using two separate mecha-nisms:(a)the mitochondria-specific form GSTK-1is expressed with an N-terminal extension which contains a putative mitochondria targeting signal[10]and(b)in the other cases the nearly intact and unprocessed GST proteins are translocated to mitochondria using signal sequences resident within the protein.The transport of nearly intact GST proteins to the mitochondrial compartment requires the help of chaperones and mitochondrial membrane-bound translocases.The mechanism of import of GSTs into mitochondria will be discussed later.
Mitochondrial GSH pool and its regulation
Despite its exclusive synthesis in the cytosol[11],GSH is distributed in other intracellular organelles.Almost 85–90%of cellular GSH is present in the cytosol, 10%–15%in the mitochondria(equivalent to10–12m m,considering the volume of mitochondrial matrix)and a small percentage is in the endoplasmic reticulum(ER) and nucleus[11].GSH exists in the reduced thiol(GSH) and oxidized disulfide(GSSG)forms.The compartmen-talization of GSH constitutes distinct redox pools in terms of balance between oxidized and reduced forms and their turnover rates.Cytosolic GSH(cGS
H)has a rapid turnover of2–3h while mitochondrial GSH (mGSH)has a relatively longer half-life of30h.GSH is predominantly( 98–99%)found in the reduced form in most cell compartments,with the exception of the ER where it exists mainly in the oxidized form,GSSG.
A shift in this balance is a good indicator of cellular redox stress.
Mitochondria are the primary site of oxygen metab-olism and their proper function is closely linked to maintenance of the GSH pool.The mGSH pool is exclusively derived from the cytosol since these organ-elles lack the enzyme system for GSH synthesis.The cytosol to mitochondrial translocation of GSH is dependent on a functional transport system.At physio-logical pH7.4,GSH exists as an anion and hence is permeable to cross the outer mitochondrial membrane. In liver and kidney mitochondria,there exist two major anion carriers,dicarboxylate and oxoglutarate, along with other anions(glutamate,citrate,aspartate, tricarboxylate)which mediate the exchange of GSH for phosphate and dicarboxylate across the inner mitochondrial membrane.The uptake of GSH by rat kidney mitochondria appears to be saturable(K m= 1.3m m,V max=5.59nmolÆmin)1Æmg)1protein).How-ever,it is not clear if this is a general mechanism of GSH import in all tissues[11,12].Mitochondrial mem-branefluidity,especially cholesterol content,is also a key factor regulating GSH import into mitochondria. Increase in cholesterol content of the inner mitochon-drial membrane,as seen in rodents
under chronic treat-ment with alcohol or under certain oxidative stress conditions such as hypoxia,diminishes GSH import into mitochondria.It is noteworthy that GSH trans-port to mitochondria is unidirectional since no back transport of mGSH to the cytosol has been reported. Increasing thefluidity of the mitochondrial inner mem-brane increases cGSH transport into mitochondria [12,13].
mGSH pool in pathology
mGSH status is closely associated with mitochondrial oxygen consumption,reactive oxygen species(ROS) production and redox status.A majority of mitochon-drial respiratory and transport enzymes contain critical sulfhydryl groups that must be maintained in the
Dual localization of glutathione transferase H.Raza
reduced form.Alteration in mGSH concentration has been associated with numerous oxidative stress related disorders including aging,cancer,diabetes,hypoxia, ischemia⁄injury and other diseases associated with cardiac,hepatic and neurological functions[12–15]. Under experimental or pathophysiological conditions, mGSH and cGSH pools can be selectively depleted, and upon depletion recovery of the mGSH pool takes a significantly longer time[10,13]than that of cGSH. Our previous studies have also shown that in cells trea-ted with an oxidant lipid aldehyde,4-hydroxynonena
l (4-HNE),the cGSH and mGSH pools are differently affected and the recovery of the mGSH pool is signifi-cantly delayed compared with the cGSH pool[8].The mGSH pool size is also dependent upon the turnover and metabolism of GSH in mitochondria[12,14]. Under oxidative stress conditions,as seen in ischemia-associated cardiovascular and neurological disorders, altered mGSH content and the ratio of mGSH to GSSG are directly associated with increased produc-tion of ROS[12–15].Therefore,a sustained mGSH pool in the mitochondrial matrix would be advanta-geous to minimize the potential ROS-induced oxidative insults during physiological and pathophysiological metabolism of oxygen in the mitochondria[15].
Under both physiological and pathological condi-tions,the mGSH pool is regulated by a combination of mGSH transport activity and GSH metabolism. The important enzymes of GSH metabolism inside the mitochondria are glutathione peroxidases(GSHPX1 and4)which protect(detoxify the endogenous and exogenous toxic peroxides by conjugating with GSH) the mitochondria against oxidative degeneration.The other GSH metabolizing enzymes are mGSTs,which play a significant role in protecting mitochondrial functions by enzymatic transfer of GSH to proteins and metabolites.mGSH is also utilized for glutathiony-lation of proteins by glutaredoxin-2⁄thioltransferases. mGSH reductase,which recycles GSSG back to GSH, also plays a significant role in protecting mitochondria a
gainst oxidative stress.These enzymes are differen-tially regulated in the cytosol and mitochondria when exposed to chemical,biological or physical insults [12,14–16].
mGST pool
The mGST(GST13-13)wasfirst identified in rat liver mitochondria by Harris et al.[6].This enzyme was later characterized as GST kappa(GSTK1-1)and the human (hGSTK1-1)and murine(mGSTK1-1)homologues have since been identified[10,17].Over the years,multi-ple forms of GST in hepatic mitochondria have been characterized in rats,mice and humans[6–10].How-ever,very little is known about their physiological properties,functions and regulation.Mitochondria are the main site for ROS production during respiration coupled oxygen metabolism that may ultimately dam-age membrane lipids,DNA and proteins.Conse-quently,it is believed that GSTs play a key role in protecting mitochondrial genetic and metabolic machin-ery against oxidative insults.mGSTs are also presumed to render protection against cardiolipin oxidation in the mitochondrial inner membrane,which in turn prevents the release of cytochrome c and initiation of apoptosis. We have previously reported the presence of class alpha and mu GSTs in rat and mouse liver mitochondria and also in rat brain mitochondria[7,8,18].Gallagher et al.
[9]showed the presence of alpha GSTA4-4only in the mitochondria but not in the cytosol.In contrast,we and others have shown the presence of GSTA4-4in both cytosol and mitochondria[8,17].
Our studies have shown that the preferred substrate for mGSTA4-4is4-HNE,an endogenous toxic product of lipid peroxidation under oxidative stress conditions.Our recent studies using immunolabeling and confocal microscopy have indicated increased translocation of GSTA4-4from cytosol to mitochon-dria when cells were incubated with4-HNE,tumor promoter phorbol ester(PMA),or cAMP(protein kinase A activator)suggesting its role in cancer and in protecting mitochondrial functions under oxidative stress conditions[8,19].Since4-HNE is considered as a signaling molecule,preferential metabolism of this molecule in the mitochondria may modulate mitochon-drial signaling pathways.We have also identified other GST isoenzymes,GSTA1⁄2and GSTM1⁄2,in mouse liver mitochondria[8],which was later confirmed by Thomson et al.[17]and in human liver by Gallagher et al.[9].Gallagher et al.[9]have also described the occurrence of trace amounts of GSTPi in the human liver mitochondria.A recent study by Goto et al.[20] has shown the presence of GSTPi in the mitochondria as well as nucleus of mammalian cells.
Mitochondrial targeting of GSTs
Several studies have shown that GST isoenzymes in mitochondria are structurally and catalytically similar to their cytosolic counterparts[6–9,17–20].These pro-teins are now often termed‘echoproteins’.These studies indicate that the import of mGST isoforms depends on the internal cryptic signals,without any proteolytic processing or alternative translation of the protein,sim-ilar to what has been established for some of the mito-chondrial cytochrome P450s(CYPs)and other proteins
H.Raza Dual localization of glutathione transferase
[21–23].Mitochondrial-specific GST kappa(GSTK1-1), however,is a unique GST,distinct from the cGSTs as it has a putative cleavable N-terminal signal for mitochondrial translocation and a C-terminal signal sequence,Ala-Arg-Leu,for peroxisomal targeting[10]. In general,the majority of mitochondrial imported proteins have15–40N-terminal cleavable residues rich in positively charged and hydroxylated amino acids which form an amphipathic helix essential for interac-tion with negatively charged residues on the translocase of the outer membrane of the mitochondrial complex and import of proteins from the cytosol to the mitochondrial matrix([22]and the accompanying review by Avadhani et al.[24]).However,a number of mitochondria targeted proteins,particularly the xenobiotic inducible CYPs,lack a cleavable N-terminal pre-sequence([21,23–30]and the accompanying reviews by Knockaert et al.[31]and Yogev et al.[32]).In these cases,the bimodal targeting of proteins to ER and mit
ochondria is catalyzed by the chimeric signals they carry.Furthermore,the bimodal targeting of these predominantly microsomal CYPs is facilitated by post-translational phosphorylation by kinases under physiological and pathological conditions([25–30]and Knockaert et al.[31]in this series).Protein kinase A mediated phosphorylation of serine residues of CYPs increases the affinity of proteins for binding to cyto-plasmic chaperones such as heat shock proteins(Hsp), Hsp70⁄Hsp90,resulting in increased mitochondrial translocation[28–30].reactive oxygen species是什么意思
The molecular mechanism by which recombinant mouse mGSTA4-4is targeted to mitochondria was investigated using a combination of in vitro mitochon-drial import assay and in vivo targeting in COS cells transfected with GSTA4-4cDNA.Results showed that mGSTA4-4is hyperphosphorylated compared with cGSTA4-4.Both cAMP and PMA markedly increased the import of GSTA4-4from cytosol to mitochondria. Mutational analysis shows that the putative mitochon-drial targeting signal in GSTA4-4resides within C-ter-minal20amino acids and Ser189and Thr193are the sites for phosphorylation activation of the import sig-nal[25].The targeting function of the C-terminal sequence was further confirmed in experiments show-ing that the C-terminal172–222sequence of GSTA4-4 was able to target the N-terminally fused,but not C-terminally fused,dihydrofolate reductase(a cytosolic protein)to mitochondria.In addition,we have also provided evidence that hyperphosphorylated mGST-A
4-4has an increased affinity for molecular chaperone Hsp70compared with the hypophosphorylated cGSTA4-4.Our hypothesis is that the newly synthe-sized GSTA4-4subunits have two fates:(a)cyto-solic retention due to inefficient phosphorylation and Hsp70binding which results in rapid folding and dimerization of subunits which makes them incompetent for import;(b)mitochondrial import due to hyperphosphorylation and Hsp70binding prevent-ing rapid dimerization in the cytosol and making them import competent[25](Fig.1).The import competent conformational change in GSTA4-4is augmented under oxidative stress conditions,due to increased ROS production as seen in numerous diseases,suggest-ing a physiological role of mGSTA4-4(Fig.2A,B).
O2.
LPO
HNE O2.–
GSH GSSG
GST –
Dual localization of glutathione transferase H.Raza
In a recent study,Goto et al.[20]have reported that mitochondrial targeting of GSTPi also involves no detectable protease processing.They observed no dif-ference in the size of cGSTPi and mGSTPi forms and the mitochondrial translocation depended on an inter-nal signal located at its N-terminal region.They identi-fied two clusters (1–19and 71–84)of positively charged amino acid rich regions as possible mitochon-dria targeting signals of mGSTPi.The reasons for the observed difference in the location of signal from C-terminal in the case of GSTA4-4to N-terminal in the case of GSTPi remain unclear.Nevertheless,it is clear from these studies that cryptic mitochondria tar-geting signals of GST isoforms are used for their bimodal targeting to mitochondria.Physiological role of mGSH and GSTs
ROS production in the mitochondria at the sites of complex I and complex III of the respiratory chain is a
physiological process occurring during oxygen reduc-tion and ATP synthesis.ROS at a lower level may function as signaling intermediates for cell survival [33].ROS at higher level cause oxidative stress and cell death and have been implicated in the pathogenesis of many diseases,notably neurodegeneration,aging,can-cer,ischemia and diabetes [33,34](Fig.2A,B).GSH is critical for cellular functions,cell growth and cell death.GSH also regulates signaling pathways mostly by maintaining redox status,ROS,sulfhydryl groups of cellular proteins,and oxidative stress which activates various sig
nal transduction and transcrip-tional pathways [10,12,15,16,23].As mentioned above,mitochondria lack GSH synthesis and therefore depend on the import of cGSH.Increased cellular GSH promotes the growth of normal as well as cancer cells presumably by modulating rate-limiting enzymes in DNA synthesis and cell cycle progression [10,12,35].GSH also modulates cell death by redox regulation of mitochondrial functions,ATP synthesis and thiol con-tents of signaling molecules like NF j B,stress kinases and caspases [10,12,35].
As mentioned above,the mGSH pool (10–15%)is metabolically separate from the cGSH pool in terms of synthesis and turnover.Increased mitochondrial stress has been reported when the mGSH level is below a critical level (i.e.2–3nmol Æmg )1protein)[15].Recent studies have shown that mGSH also plays a significant role in trafficking antiapoptotic Bcl-2and proton transporter uncoupler protein 2(UCP2)and therefore is critically involved in cell survival and cell death mechanisms [15].Additionally,mGSH also interacts with NO by formation of S -nitroglutathione and thus mGSH may serve as an NO donor or reservoir.The high concentration of mGSH and mGSTs and the pres-ence of NO synthase in mitochondria suggest the physiological role of mGSH [15,35].GSH-dependent protein S-glutathionylation of mitochondrial complex I is involved in shifting the balance of mGSH ⁄GSSG physiologically and is also implicated in oxidative damage in many pathologies and altered mitochondri
al bioenergetics [36].Similarly,glutathionylation of mito-chondrial complex II has been implicated in post ische-mic heart diseases [35].Glutathionylation of adenine nucleotide translocase has been reported to prevent mitochondrial membrane permeabilization and apopto-sis [37].Furthermore,GSH contributes to the reduc-tion of physiological hydroperoxides,including the products of lipid peroxidation,through GST and glu-tathione peroxidase (GSHPX)which will be discussed later in this review.These studies have shown that mGSH plays multiple roles in maintaining mitochon-drial bioenergetics in normal and disease conditions.
mito GSH +GST pools
H 2O 2
LPO
HNE
O 2.–
GSH
GSSG
OH .
O H O O -.e –e –
e –
OH .e –2H O
O 2
2H 2O O 2
O 2–
.
>90%(ROS)
1 e –
~1-2%Mitochondrial Respiration
+ATP
4e–4H +
leak
A
H.Raza Dual localization of glutathione transferase

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