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Molecular & Biochemical Parasitology 197 (2014) 1–8

Contents lists available at ScienceDirect

Molecular & Biochemical Parasitology

eview

ndoplasmic reticulum stress responses in Leishmania

ubhankar Dolai1, Subrata Adak ∗

ivision of Structural Biology & Bio-informatics, CSIR-Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Kolkata 700 032, India

r t i c l e i n f o

rticle history:eceived 5 March 2014eceived in revised form 14 August 2014ccepted 5 September 2014vailable online 16 September 2014

eywords:nfolded protein responseR quality controlRADutophagyell death

a b s t r a c t

Perturbation of endoplasmic reticulum (ER) homeostasis can lead to an accumulation of misfolded pro-teins within the ER lumen causing initiation of ER stress. To reestablish homeostasis and mitigate thestress, a series of adaptive intracellular signaling pathways termed the unfolded protein response (UPR)are activated. ER stress is of considerable interest to parasitologists because it takes place in parasites sub-jected to adverse environmental conditions. During a digenetic lifestyle, Leishmania parasites encounterand adapt to harsh environmental conditions that provide potential triggers of ER stress. These includenutrient deficiency, hypoxia, oxidative stress, changing pH, and shifts in temperature. Protozoan humanpathogens, including the causative agents of trypanosomiasis, leishmaniasis, toxoplasmosis and malaria,contain a minimal conventional UPR network relative to higher eukaryotic cells. Three different signalingpathways in the ER stress response have been described in trypanosomatids: these pathways involve (i)the down-regulation of translation by a protein kinase RNA-like ER kinase (PERK), (ii) the ER-associateddegradation (ERAD) pathway, and (iii) the spliced leader silencing (SLS) pathway and its target mRNAs.

Under short-term ER stress, signaling from PERK activates autophagy, a cell survival response. But bothchronic and unresolved ER stresses lead to initiation of apoptotic events and eventual cell death. Thisreview presents the current understanding of the ER stress response in Leishmania with an emphasison protein folding and ER quality control, unfolded protein response, autophagy as well as apoptosis inreference to the mammalian system.

© 2014 Elsevier B.V. All rights reserved.

ontents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12. ER functions in Leishmania . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23. Protein folding, N-linked glycosylation and ER quality control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

3.1. Function of ER chaperones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33.2. Function of glycosylation/deglucosylation enzymes, calnexin/calreticulin and oxidoreductases in the ER . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

4. ER stress and autophagy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45. UPR adaptive response during ER stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56. A death response during ER stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

7. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

∗ Corresponding author. Tel.: +91 33 2473 6793; fax: +91 33 2473 5197.E-mail address: adaks@iicb.res.in (S. Adak).

1 Present address: Department of Medicine, University of Toronto, Medical Sci-nces Building, Room 7368, 1 King’s College Circle, Toronto, Ontario M5S 1A8,anada.

ttp://dx.doi.org/10.1016/j.molbiopara.2014.09.002166-6851/© 2014 Elsevier B.V. All rights reserved.

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1. Introduction

Leishmania spp. are the causative agents of a spectrum of humandiseases collectively known as leishmaniasis. The parasites alter-

nate between extracellular promastigotes in the insect vector andintracellular amastigotes within the macrophages of human host. Inthe pathogenic amastigote stage, the parasite is elaborately adaptedto escape the host immune system and to facilitate nutrient uptake

2 chemical Parasitology 197 (2014) 1–8

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Fig. 1. The key players known to be involved in ER stress homeostasis during theLeishmania life cycle. ER stress activates the stress sensors activating transcrip-tion factor (ATF) 6, inositol-requiring kinase 1 (IRE1), and PKR-like endoplasmicreticulum kinase (PERK) in higher eukaryotes. Both ATF6 and IRE1 are absent inLeishmania. PERK (via eIF2� phosphorylation) decrease the load of proteins thatmitigate ER stress. An evidence-based hypothesis in trypanosomes is that the SLS

S. Dolai, S. Adak / Molecular & Bio

rom parasitophorous vacuole of host cell. Like other eukaryotes,he endoplasmic reticulum (ER) in Leishmania parasites is the hubf the secretory pathway, where secretory or membrane proteinsre folded prior to trafficking to other sites within the secretoryetwork.

The development of proper protein-folding is achieved through coordinated interplay of modifying enzymes, molecular chaper-nes and folding enzymes with the nascent proteins within theptimized environment of the ER lumen. Correctly folded proteinsre transported to the Golgi apparatus for further maturation andistribution to their destinations. Misfolded proteins are detectedy ER quality control (ERQC) systems and are removed from the ER

umen by retrotranslocation to cytosol where they are degraded by6S proteasomes of the ER-associated degradation (ERAD) machin-ry [1]. Misfolded proteins and even damaged ER can also beemoved by autophagy [2]. The delicate process of protein folding isensitive to changes in protein glycosylation, calcium flux, temper-ture, and exposure to reducing agents. Alteration of any of thesean lead to misfolding of proteins and their accumulation within theR lumen initiating ER stress. To alleviate ER stress, cells in higherukaryotes activate a series of ‘self-defense’ signaling pathwaysollectively termed the unfolded protein response (UPR). Higherukaryotes sense an UPR by the luminal domains of three ER trans-embrane proteins: inositol-requiring kinase/endoribonuclease 1

IRE1), protein kinase RNA (PKR)-like ER kinase (PERK), and basiceucine-zipper activating transcription factor (ATF) 6 [3]. By usinghese three sensor/transducer pathways, the ER responds to mildtress through decreasing protein translation and upregulating aeries of genes encoding proteins involved in ER protein fold-ng, ERAD, ERQC, autophagy and lipid biogenesis. In multi-cellularukaryotes subjected to prolonged ER stress, cells induce apoptosiso safely remove injured cells to ensure organism survival [3]. Sur-risingly, Ire1 is also present in Schizosaccharomyces pombe where

t contributes to ER homeostasis through two post-transcriptionalechanisms, rather than regulation of the transcriptional machin-

ry: during the UPR, it initiates Ire1-dependent mRNA decay (RIDD)f a large, select set of ER-targeted mRNAs and processes Bip1 (aajor ER chaperone) mRNA that escapes decay, thereby stabilizing

t [4].Trypanosomatids, including Leishmania, are highly divergent

rom higher eukaryotes due to their lack of conventional trans-riptional regulation [5], which includes a lack of RNA polymeraseI promoters for protein-coding genes. Trypanosomatids also lackRE1 and ATF6, which act along the transcriptional regulatoryranches of the UPR (Fig. 1). In contrast in silico and func-ional data indicate a single identifiable UPR sensor related tohe PERK is present in trypanosomatids, which acts on the reg-lation of protein translation [6]. In addition, trypanosomatidsontain both a complex ERQC system and an apparently simplifiedRAD machinery to monitor protein folding and the degradationf misfolded proteins, respectively [7]. Other human pathogensToxoplasma gondii and malarial parasites) are also thought to lack

transcriptional response to the UPR, and consequently in theses well as trypanosomatid parasites, regulation at the transla-ional level and identification/disposal of misfolded ER proteinsepresent major compensatory mechanisms in parasites to main-ain ER homeostasis during an UPR (Fig. 1) [8–11]. Recently oneroup of workers hypothesized that a post-transcriptional pro-ram, the spliced leader silencing (SLS) pathway, is elicited uponR stress in Trypanosoma brucei, and is used by the parasites as

mechanism to accelerate the cell death and to thus, eliminatenfit organisms from the population [12,13]. Here, we review ER

tress response in the lower eukaryotes Leishmania as well ashe higher eukaryotes mammals in every context to facilitate thenderstanding of similarities and discrepancies between two sys-ems.

pathway is induced by ER stress via PK-3, a serine-threonine kinase. SLS leads toa complete shutdown in trans-splicing, resulting in reduction in mRNA productionand to induction of cell death.

2. ER functions in Leishmania

Like mammals, the ER of Leishmania constitutes an endomem-brane compartment within the cell that is organized byinterconnected tubules and sacks that extend throughout the cellbody. The main functions of ER include biogenesis, modificationsand transportation of secretory proteins, synthesis of lipids, andstorage of Ca2+ [14]. For instance, within the Leishmania ER fattyacids are synthesized de novo by a fatty acid elongation (FAE)pathway comprising elongase 1 to elongase 4 (Elo1-4) [15] andthe ER is also the site of synthesis for glycolipids, the glyco-sylphosphatidylinositol (GPI) anchors attached to many plasmamembrane-exposed proteins, and another key surface componentlipophosphoglycan (LPG). Research indicates that GPI anchors areadded to the C-terminus of proteins carrying a GPI anchor attach-ment signal within the ER lumen, and this represents an importantpost-translational modification in the context of abundant surfaceglycoproteins that are central to the virulence of trypanosomatidparasites [16]. These proteins include the protease gp63 in Leish-mania, the procyclic acidic repeatitive proteins (procyclins) andvariant surface glycoproteins (VSG) of Trypanosoma brucei, and the1G7 antigen of Trypanosoma cruzi. GPIs also provide the anchor-ing determinant for the cell surface lipophosphoglycans (LPGs) andglycoinositolphospholipids (GIPLs) [16]. Collectively, GPI anchoredsurface-exposed proteins and glycolipids contribute to a protec-tive shield that is crucial for Leishmania survival in the insectgut and within macrophages of the human host [17]. Another ER

dependent crucial posttranslational modification required for pro-tein folding, N-glycosylation, is discussed in Section 3. The ER alsoplays a major role for intracellular Ca2+ storage and signaling ineukaryotic cells [18]. ER localized sarco-endoplasmic reticulum

S. Dolai, S. Adak / Molecular & Biochemical Parasitology 197 (2014) 1–8 3

Table 1Comparative studies of protein folding and quality control in the ER between mammals and kinetoplastids. Dgt1, EDEM, ERGIC-53, Lag1, OS9 and UGGT are delayed GPI-anchored protein transport, ER degradation-enhancing �-mannosidase-like protein, ER-Golgi intermediate compartment, longevity assurance gene 1, Osteosarcoma 9, andUDP-glucose-glycoprotein glucosyltransferase, respectively.

Proteins and chaperones Functions in mammals Status/functions in Kinetoplastids

Sec61 Part of ER translocation channel Orthologue is present

BiP/GRP78 Assists proteins translocation and folding in the ER. Assistsretrotranslocation of misfolded proteins and ERAD. RegulatesUPR by interacting with IRE1, ATF6 and PERK [25]

Assists proteins translocation and folding in the ER [26]

Endoplasmin/GRP94/LPG3 Secretory pathway chaperone Orthologue is present (Tb927.3.3580)

DNAj BiP co-chaperone Orthologue is present [8]

Glucosidase I Removes 1st glucose from N-linked glycans [36] Lacks glucosidase I [7,93]

Glucosidase II Removes both 2nd glucose and 3rd glucose from N-linkedglycans [36]

Glucosidase II removes UGGT incorporated glucose andthought to terminate calreticulin folding cycle [7]

Calnexin and calreticulin Specifically binds to monoglucosylated glycans leading to theirassociation with glycoproteins [36]

Express only calreticulin [7]

PDI, ERO1 and ERp57 Disulfide bonds formation/rearrangement oncalnexin/calreticulin bound glycoproteins. Removes disulfidebonds and facilitate ERAD of misfolded proteins [36]

Orthologues of PDI, ERO1 and ERp57 genes are present [44]

UGGT Reglucosylates incompletely folded glycoproteins tomonoglucosylated glycoproteins that re-associate them withcalnexin/calreticulin [36]

Adds glucose residue to trypanosome N-glycan(Man6–7GlcNAc2) [41]

�-Mannosidase I Removes one of the 9 Man and targets the glycoproteins toEDEM [36]

Lacks �-mannosidase I [7]

EDEM Binds to �-manosidase I cleaved 8 Man containingglycoproteins and target them for ERAD [36]

Orthologue of EDEM gene is present in Trypanosoma butunknown in Leishmania [7]

OS9 Specifically binds to unfolded glycoproteins with 8/5 Mancontaining glycans [36]

Orthologue of OS9 gene is present [7]

Lag1/Dgt1 Transport GPI-anchored proteins from ER to Golgi Orthologues of Lag1/Dgt1 genes are present

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ERGIC-53 Lectin; binds to Man residues on N-glycans oproteins and transport them to Golgi

a2+-ATPase (SERCA), and intraluminal Ca2+-binding proteins con-ribute to the Ca2+storage from 100 �M to 5 mM. However, otherR localized proteins: Ca2+ release channels, ryanodine receptorsRYRs) and InsP3 receptors (InsP3Rs) contribute to the regulation ofntracellular Ca2+ release upon activation. The Leishmania genomeatabase reveals the presence of candidate SERCA (LmjF.04.0010),YR/InsP3R (LmjF.16.0280) orthologues. The ER localized SERCA

n L. amazonensis has been shown to regulate parasite virulencehrough regulation of intracellular Ca2+ [19]. The InsP3R in T. bru-ei is required for growth and infectivity and has been localized incidocalsisome, an acidic Ca2+ storage compartment [20]. The local-zation and functions of InsP3R and RYR in Leishmania are neededo be characterized.

. Protein folding, N-linked glycosylation and ER qualityontrol

Transmembrane and secretory proteins entering the ER lumenequire proper folding and their subsequent exit as eitherolded proteins in transit to their target organelles or as mis-olded proteins targeted for degradation [21]. The ER presents aighly oxidizing environment and harbors a high concentrationf Ca2+-binding/Ca2+-dependent chaperone proteins, glycosyla-ion/deglucosylation enzymes, ER oxidoreductin 1, and proteinisulfide isomerases. These proteins provide suitable milieu andssistance to nascent proteins to fold correctly in different stagesf their maturation (Table 1).

.1. Function of ER chaperones

Heat shock protein (HSP) 70 family binding protein (BiP), alsoalled glucose regulated proteins 78 (GRP78), is the most abundant

erly folded Orthologue of ERGIC-53 is present [5]

ER chaperone and first to encounter newly synthesized proteinstraversing the ER membrane through the Sec61 translocon com-plex [22]. BiP interacts with the hydrophobic domains of proteins,and facilitates their translocation, by preventing misfolding andaggregation. These functions of BiP are attributed to its peptide-dependent ATPase property. The ATPase activity of BiP is stimulatedby binding of peptide and subsequently generates the ADP-boundform that has high affinity for the bound peptide. The J-domaincontaining HSP40 family of co-chaperones ERdjs stimulate ATPhydrolysis of BiP and assist its chaperone activity by stabilizingclient binding [23]. Nucleotide exchange of ADP with ATP releasesthe substrate from BiP allowing other chaperones to interact andcontinue the folding process. Binding of BiP to ER stress trans-ducers prevents UPR signaling in mammalian cells [24]. BiP alsoassists in the delivery of soluble misfolded proteins for degra-dation [25]. HSP90 family chaperone GRP94 (Glucose regulatedproteins 94) chaperones a limited number of proteins by stabilizingadvanced folding intermediates and incompletely assembled pro-teins. It binds substrates after they have been released from BiP. Thetrypanosome genome contains BiP, three HSP70, GRP94/LPG3 andfour potential ER targeted DnaJ-domain proteins displaying fea-tures common to ERdj proteins (Tb09.211.3680, Tb 10.70.5440, Tb11.01.8480 and Tb927.3.1430) [8]. In bloodstream form T. brucei,BiP has been shown to be localized within ER and physically inter-act with secretory protein VSG [26]. In T. cruzi BiP has been shownto associate with cathepsin L (TcrCATL)/cruzipain in its early stageof folding [27], suggesting BiP’s role as a molecular chaperone intrypanosomes. In L. donovani, BiP is able to form complex with A2

protein in the ER under conditions of heat shock [28], and maytherefore prevent from misfolding at higher temperature. A Leish-mania orthologue of mammalian ER chaperone GRP94, LPG3, playsa vital role in the phosphoglycan biosynthesis implicated in parasite

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S. Dolai, S. Adak / Molecular & Bio

irulence but not viability [29,30]. However, the LPG3 mRNA is reg-lated developmentally but not by stress or heat, indicating that itsole in Leishmania differs significantly from other eukaryotes.

.2. Function of glycosylation/deglucosylation enzymes,alnexin/calreticulin and oxidoreductases in the ER

Within the ER lumen, secretory proteins are frequentlylycosylated at asparagine (N) residues in a process termed ‘N-lycosylation’. N-glycosylated proteins are folded in assistanceith calnexin and/or calreticulin lectin (carbohydrate-binding pro-

ein) chaperones [31]. For this process, oligosaccharides (glycans)re initially assembled on a polyisoprenoid lipid career dolicholDL) at the cytosolic face of the ER membrane in assistanceith a series of glycosyltransferases encoded by asparagine-linked

lycosylation genes (ALGs). First, N-acetylglucosamine (GlcNAc)-hosphate from UDP-GlcNAc is added to dolichol-phosphateDL-P) by ALG7 encoded N-acetylglucosamine-phosphate trans-erase to generate DL-PP-GlcNAc [32]. Subsequently a secondlcNAc residue and five mannoses (Man) from GDP-Man arettached to DL-PP-GlcNAc by the sequential action of ALG13/14,LG1, 2 and 11 encoded glycosyltransferases to build DL-PP-lcNAc2Man5. DL-PP-GlcNAc2Man5 is then translocated to theR lumen and further elongated by addition of four additionalan from DL-P-Man by ALG3, 9 and 12 [32]. Finally three glu-

ose (Glc) molecules from DL-P-Glc are added catalyzed by ALG6, and 10 to build DL-Glc3 GlcNAc2Man9, which is then trans-erred en bloc by the catalytic subunit STT3 of hetero-oligomericnzyme oligosaccharyltransferase (OST) to the amide side chainf an Asn within the consensus Asn-X-Ser/Thr (X is any aminocid except proline) sequence of nascent polypeptide chains [33].onoglucosylated glycoproteins are formed from Asn-linked gly-

ans by subsequent removal of two Glcs by glucosidase I andlucosidase II [34] and specifically recognized by the lectin domainsf soluble calreticulin or the membrane-anchored calnexin [34].inding by calnexin and/or calreticulin retains the glycoprotein

n the ER lumen allowing oxidoreductases like protein disulfidesomerase (PDI) and calnexin/calreticulin bound ERp57 to formisulfide bonds through oxidation of cysteine residues of nascentroteins [34]. PDI also rearranges incorrect disulfide bonds as an

somerase, prevents aggregation of unfolded nascent proteins as molecular chaperone, and removes disulfide bonds from ter-inally misfolded proteins to facilitate ERAD [1]. Reduced PDI is

eoxidized by ER oxidoreductin (ERO1) where molecular oxygenO2) acts as a terminal electron acceptor [35]. Removal of the thirdnd last Glc by glucosidase II terminates the interaction of gly-oproteins with calnexin and/or calreticulin. If, however, foldingf glycoproteins is not successful, UDP-glucose: glycoprotein glu-osyltransferase (UGGT) re-glucosylates the oligosaccharide chainnd allows another folding attempt by associating with calnexinnd/or calreticulin. The cycle continues until the protein foldedroperly. Failure in refolding results trimming of single Man fromlycan by ER �1–2 mannosidase I from the middle branch of gly-an and subsequently the release of the unfolded proteins fromalnexin and/or calreticulin is occurred. The unfolded proteinsith 8 Man containing glycans are recognized by ER degradation-

nhancing 1,2-mannosidase-like protein (EDEM) or osteosarcoma (OS9) lectin proteins that target the unfolded proteins to retro-ranslocon complex, leading to ERAD [36].

Many secretory proteins in Leishmania, including the gly-oprotein gp63 and proteophosphoglycans (PPGs) undergo N-lycosylation. This is an important for Leishmania survival as

nhibition of N-glycosylation by tunicamycin or deletion of theene encoding glutamine: fructose-6-phosphate amidotransferasenvolved in N-glycan synthesis results growth arrest and loss ofirulence [37]. The biosynthetic pathways of N-glycans are similar

ical Parasitology 197 (2014) 1–8

to higher eukaryotes. However, Leishmania differ from their humanhosts by synthesizing shorter DLs that contain 11–12 isoprene unitsin comparison to 18–21 units in mammals [38]. Leishmania alsolack several homologues of ER luminal glycosyltransferases likeALG12, 6, 8 and 10; and thus synthesize small non-glucosylatedDL-linked glycans with 6–7 Man residues (DL-Man6–7GlcNAc2)[7]. Kinetoplastids are also unique by encoding only the STT3 cat-alytic subunit homologues of OST. T. brucei and L. major encodes 3and 4 paralogs of STT3, respectively, with distinct preferences fortheir polypeptide substrate and can complement yeast OST com-plex individually independent of other yeast OST subunits [39,40].Unlike yeast and mammalian OSTs that are selective to DL-Glc3GlcNAc2Man9, trypanosome STT3s can transfer different lipid linkoligosaccharides at the same rate, a possible strategy to glycosylatea different array of proteins with different oligosaccharides [40]. InLeishmania N-linked glycans are glucosylated by one or two glucoseresidues by Leishmania encoded UGGT [41]. Trypanosomes encodeonly calreticulin [7] that has been shown to interact specificallywith monoglucosylated lysosomal protein cruzipain/TcrCATL in itsadvanced state of folding [27,42]. Overexpression of independentlyfolding P domain of calreticulin in L. donovani results the inhibitionof the parasite secretory acid phosphatases [43] indicating that thealteration of the function of calreticulin in Leishmania may affectthe targeting of proteins. Mutation of the cysteine residues in ERlocalized single CGHC motif containing L. donovani PDI results inthe loss of both oxidase and isomerase activities in vitro as well assecretory inhibition in vivo [44]. So, Leishmania calreticulin and PDIappear to play an important role in the ERQC. Glycans are deglu-cosylated by glucosidase II as glucosidase I is absent in Leishmania[7]. Bioinformatics-led studies reveal the presence of yeast Yos9homologue protein in Leishmania [7]. In yeast, lectin protein Yos9(yeast homologue of osteosarcoma 9) performs similar functionsto EDEM in the ERAD pathway [45]. However Yos9 detects glycanswith 8 or 5 Man residues [45], whereas Leishmania glycans contain6–7 Man residues, indicating requirement of further investigationsto understand the mechanism of releasing unfolded proteins fromcalreticulin and their degradation.

4. ER stress and autophagy

Autophagy serves as a cytoprotective mechanism through thelysosome-dependent recycling of cellular material including dam-aged organelles and protein aggregates. In apoptosis-resistantmammalian cells, autophagy has also been classified as a formof programmed cell death [46]. An elevated level of autophagy isobserved in ER stressed cells to mitigate stress by removing aggre-gated and misfolded proteins in conjunction with the arrest ofgeneral protein synthesis and ERAD [47]. IRE1, PERK and increasedcytosolic Ca2+ have been implicated as mediators of ER stress-induced autophagy in mammals [48]. The activated kinase domainof IRE1 recruits tumor necrosis factor (TNF) receptor-associatedfactor 2 (TRAF2) and activates c-Jun N-terminal kinase (JNK)pathway through mitogen-activated protein kinase kinase kinase(MAPKKK) and apoptosis signaling-regulating kinase (ASK1). Thedownstream mechanism of autophagy is thought to be a resultof JNK mediated phosphorylation of B-cell leukemia/lymphoma 2(Bcl2) resulting its dissociation from Beclin1 to activate autophagy-inducing class I phosphoinositide (PI) 3-kinase PI3K/vacuolarprotein sorting (Vps) 34 [49]. Activation of PERK has been shown tomediate the transcriptional activation of the proteins microtubule-associated protein 1 light chain 3 (LC3/Atg8) and autophagy-related

protein (Atg) 5 through eIF2�-ATF4-CHOP axis of signaling path-way under hypoxia. Activated PERK mediated translational blockof inhibitor of nuclear factor (NF)-�B (I�B) and thereby activa-tion of NF-�B, which could also contribute to autophagy [49].

chemical Parasitology 197 (2014) 1–8 5

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Fig. 2. Adaptive and apoptotic ER stress signaling pathways in higher eukaryotes.The ribonuclease activity of IRE1 results in production of sXBP1, which inducesexpression of genes involved in restoring protein folding or degrading unfolded pro-teins. IRE1 also leads to selective degradation of mRNAs encoding membrane andsecreted proteins by RIDD. Activated ATF6 regulates the expression of UPR and ERADgenes. Activation of PERK results in suppression of global mRNA translation, withthe exception of only some mRNAs, e.g. ATF4. ATF4 induces expression of genesinvolved in restoring ER homeostasis. Activated IRE1 binds TRAF2 and activatessequentially ASK1, JNK and downstream genes responsible for autophagy and apo-ptosis. ATF4, XBP1 and ATF6 promote expression of CHOP, which transcriptionallyregulates expression of apoptosis-promoting genes. Release of Ca2+ into the cytosolstimulates autophagic and apoptotic pathways. Orthologues of shaded genes arepresent in kinetoplastids. However, their regulation and exact functions in kineto-plastids under ER stress have been subjected to investigation. See text for details.Many calpains are present in trypanosomatids but no evidence for specific calpainrequired in the cell death pathway. Abbreviations: sXBP1, Spliced form of transcrip-tion factor X-box-binding protein 1; RIDD, regulated Ire1-dependent decay; ERAD,ER-assisted degradation, TRAF2, TNF receptor-associated factor 2; ASK1, apoptosissignal-regulating kinase 1; JNK, Jun N-terminal kinase; CHOP, C/EBP homologousprotein; BIM, B-cell lymphoma 2 interacting mediator of cell death; Bcl2, B-cell lym-phoma 2; GADD 34, growth arrest and DNA damage protein 34; PDI, Protein disulfideisomerase; EDEM, ER degradation-enhancing �-mannosidase-like protein; IP3R, ER-resident inositol trisphosphate receptors; DAPK, death-associated protein kinase;CaMKK�, calmodulin dependent kinase kinase-beta; PKC�, Protein kinase C theta,AMPK, AMP activated protein kinase; mTORC1, Mammalian target of rapamycincomplex 1; BAD, Bcl2-associated death promoter; BAX/BAK, Bcl2-associated X pro-tein/Bcl2 antagonist/killer-1; CytC, cytochrome c; AIF, Apoptosis inducing factor;

their expression resulting in increase of ER chaperone and protein

S. Dolai, S. Adak / Molecular & Bio

n addition, ER-stress leads to release of Ca2+ from the ER intohe cytosol, which can activate various kinases and proteasesossibly involved in autophagy signaling. ER stress mediatedctivation of Ca2+-calmodulin dependent protein kinase kinase/beta (CaMKK2/CaMKK�) activates AMP activated protein kinaseAMPK) [49], which inhibits mammalian target of rapamycin com-lex 1 (mTORC1) and activates uncoordinated-51-like kinase 1/2ULK1/2) to promote autophagy [49]. Ca2+ also activates death asso-iated protein kinase 1 (DAPK1), a Ser/Thr kinase, in response toR stress. Activated DAPK1 phosphorylates and sequesters Beclin1rom Bcl2 resulting autophagy upregulation [49].

Autophagy has been shown in all kinetoplastid parasites,ncluding Leishmania, where it performs crucial role in cellu-ar differentiation and regulated cell death [50–52]. Genomicnd bioinformatic analyses indicate the presence of severaleast/mammal Atg related genes and their isoforms (2 fortg4: Atg4.1, Atg4.2 and 25 for Atg8: one copy canonical Atg8,

copies Atg8A, 8 copies Atg8B, 13 copies Atg8C) in Leish-ania with their possible involvement in different stages of

utophagosome maturation [53]. The biogenesis and maturationf autophagosomes in Leishmania involve Atg8 and Atg5-Atg12onjugation pathway, similar to mammals [54,55]. However inontrast to mammals where all the eight copies of Atg8s arenvolved in autophagy [56], Leishmania requires only canonicaltg8 and Atg8As for autophagosome formation [57]. Unlike mam-als where single Atg4 deals with the processing of Atg8s for

hosphatidylethanolamine (PE) lipidation/conjugation and finaleconjugation from mature autophagosomes, the processing ofanonical Atg8 at the C-terminal glycine residue and subsequentipidation to PE is mediated by Atg 4.1. The processing and con-ugation of Atg8As to PE as well as the deconjugation of bothanonical Atg8 and Atg8As from mature autophagosomes are medi-ted by Atg 4.2 [57]. In L. major, autophagosomes have been showno collect the membrane and membrane lipid PE from mitochon-rial membranes. Autophagosome formation under ER stress haseen observed in T. brucei parasites lacking IRE1 [12]. Inhibitionf autophagy by the deletion of Atg5 genes have been shown tonfluence mitochondrial dysfunction and cellular differentiationn L. major [55]. Antimicrobial peptides have been reported toause autophagic cell death in parasitic protozoa [51]. However,s autophagy has an important role in stress induced cells, the facthat autophagy activation reflects an attempted survival mecha-ism rather than induction of a cell death pathway is controversial

n this instance [58]. Ca2+-calmodulin dependent protein kinaseinase 2/beta (CaMKK2/CaMKK�) are uncharacterized in the con-ext of autophagy within kinetoplastids but T. brucei homologs ofhe � and � subunits of AMP activated protein kinase (AMPK) [59]nd the autophagy regulatory target of rapamycin (TOR) kinases areresent. Silencing TbAMPK� and TbAMPK� genes triggers changes

n surface molecule expression, and the localization of the scaffold�) subunit in glycosome suggests that it may have a role as anntermediary between surface molecule expression and glycolysis59]. Four TOR kinases are encoded in the T. brucei genome (TbTOR1,bTOR2, TbTOR3 and TbTOR4) [60,61] while three TOR kinases areeported in the L. major (LmTOR1, LmTOR2, and LmTOR3) [62].bTOR1 controls cell growth by regulating cell cycle, nucleolustructure, and protein synthesis, whereas TbTOR2 coordinates cellolarization and cytokinesis. Although TbTOR3 is involved in theontrol of acidocalcisome and polyphosphate metabolism [63], theunction of TbTOR4 plays a crucial role in the T. brucei life cycle.. major TOR3 (LmjTOR3) has been implicated in acidocalsisomeiogenesis and animal infectivity whereas the functions of Lmj-OR1 and LmjTOR2 are unclear. RNAi mediated knockdown of

bPI3K (Vps34) in T. brucei results exocytic defects and its role inxocytic protein transport has been suggested [64]. However theomplete mechanism for the induction of TOR kinases dependent

EndoG, endonuclease G; MPT, Mitochondrial permeability transition; Apaf1, Apop-totic protease activating factor 1.

autophagy in kinetoplastid parasites during ER stress is yet to beestablished.

5. UPR adaptive response during ER stress

Sequestration of BiP association or direct binding of mis-folded proteins in the luminal domains activates three arms ofER stress sensor/transducers to transmit downstream signals bytheir cytosolic domains [3] (Fig. 2). The first arm IRE1 is a type Itransmembrane protein with both Ser/Thr kinase and endoribonu-clease domains at its cytosolic face. Activation of IRE1 results inhomo-dimerization and trans-autophosphrylation, which leads tothe activation of its endoribonuclease domain. Activated endori-bonuclease domain excises a 26-base intron from mRNAs encodingbasic leucine zipper (bZIP) family transcription factor X-box bind-ing protein-1 (XBP1) [65]. Spliced XBP1 mRNA (sXBP1) translatesa more stable and active form of XBP1, which binds to ER stressresponse element (ERSE/CCAAT(N9)CCACG) of genes encoding ERresident chaperones like BiP, GRP94 and calreticulin to upregulate

folding activity [66]. The IRE1/XBP1 pathway also promotes ERAD ofmisfolded proteins and the production of phosphatidylcholine thatis accompanied by the expansion of ER membrane, hence reducing

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R stress [67]. IRE1, through its RNase activity, also degrades aubset of ER-associated mRNAs, a process known as regulated IRE1-ependent mRNA decay (RIDD) and a reduced ER protein load inammalian cells [68].The second arm of the mammalian UPR, ATF6, is a bZIP family

ranscription factor that upon ER stress translocates to the Golgipparatus where it is processed to its active form and released tohe cytosol through subsequent cleavage by site-1 (S1P) and site-2S2P) proteases [69]. Once activated, the cytosolic ATF6 migrates tohe nucleus where it can act independently or synergistically withXBP1 to induce UPR target genes like GRP78, PDI and EDEM.

Third arm PERK is a Ser/Thr protein kinase and its activa-ion leads to dimerization and trans-autophosphorylation followedy activation of its cytosolic kinase domain [3]. Activated PERKhosphorylates the �-subunit of eukaryotic translational initiationactor 2 (eIF2�) at Ser51 that leads to inhibition of mRNA transla-ion as well as attenuation of global protein synthesis and decreaseR lumenal protein burden. However genes like transcription fac-or ATF4 carrying the internal ribosomal entry site [70] bypasseshe eIF2�-dependent translational block. ATF4 promotes cell sur-ival by inducing antioxidant genes hemeoxygenase 1, glutathione-transferase and genes involved in amino-acid metabolism androtein secretion.

Leishmania parasites, which lack both the IRE1-XBP1 and theTF6 branches of UPR but elevated expression of BiP, the primearker of UPR is observed in both Trypanosoma and Leishmania

fter treatment with reducing agent DTT or protein glycosylationnhibitor tunicamycin (TM) [12,71]. Thus, does the UPR take placen these parasites, and to what extent does it differ from the path-

ay as understood in other organisms such as mammals and yeast.n absence of transcriptional regulation, UPR might be operatedy translational regulation in Leishmania. Recently a homologue ofhe ER stress inducible PERK kinase from L. infantum was charac-erized [6] suggesting that it is an ER transmembrane protein andas been shown to sense ER stress through its N-terminal lumi-al domain, and subsequently undergoes auto-phosphorylation.ctivated PERK can phosphorylate eIF2� at threonine 166 and haseen shown to be crucial for differentiation of promastigotes tomastigotes [6]. However its direct roles on translational inhibi-ion during the UPR are yet to be tested. The T. brucei homologuef PERK (TbeIF2K2) has been shown to phosphorylate T. brucei’sIF2� in vitro. However, TbeIF2K2 is located in flagellar pocket andndosomal compartment, suggesting its diverse functions withinhe parasite family [6,72]. The data of other human pathogensthe apicomplexan parasite Toxoplasma gondii) show that activatedERK can phosphorylate eIF2� and subsequently the rate of trans-ation initiation decreases [10]. In addition, approximately 500 T.ondii genes, including a subset of AP2 transcription factors andther transcription modulators, showed enhanced association withranslating ribosomes during ER stress [10]. Thus, the availablevidence in intracellular parasites suggests that translational reg-lation seems to have a central role in responding to stress.

In higher eukaryotes, processing (P)-bodies add another levelf translational control by degrading mRNA and attenuatingranslation through eIF2� phosphorylation under stress [73]. Inrypanosomes, sequestration of mRNAs in stress granules (SGs) and-bodies under stress is accompanied by increased mRNA turnover,hich is independent on eIF2� phosphorylation [74]. However,

hese data suggest that both SGs and P-bodies could provide andditional adaptive mechanism in trypanosomes during stress buturther experimental evidence is required to understand how thisorks.

In mammals, Ca2+ dependent phosphatase calcineurin has beenroposed to play both protective and apoptotic roles upon ER stress.alcineurin has been shown to interact with PERK and stimu-

ate autophosphorylation, and eIF2� mediated translational block

ical Parasitology 197 (2014) 1–8

[75]. Deletion of Ca2+ binding subunit of calcineurin in Leishmaniarenders cells susceptible to TM induced ER stress [76]. However,downstream target molecules need to be identified.

6. A death response during ER stress

Persistent and unresolved ER stress leads to apoptosis (Fig. 2). Inhigher eukaryotes, ER stress-induced cell death is mediated by allthree branches (PERK, IRE1 and ATF6) of UPR signaling through theproduction of common death mediator C/EBP homologous protein(CHOP), a bZIP family transcription factor. The switching to apopto-sis is thought to be the result of surplus production and availabilityof the apoptotic mediator CHOP and its downstream product, thegrowth arrest and DNA damage-inducible protein 34 (GADD34) [3].CHOP-induced apoptosis leads to the reduction in Bcl2 pro-survivalprotein expression and an increase in pro-apoptotic proteins likeBIM, PUMA and BAX [77,78]. GADD34 dephosphorylates eIF2�,resulting in the removal of translational repression and an increasein ER protein load and apoptosis [79]. CHOP also induces ER oxi-doreductin (ERO1) which in turn increases ER oxidative stress andpromotes the release of ER-stored Ca2+ to the cytosol throughIP3R1 [80]. The elevated cytosolic Ca2+ concentration subsequentlyinduces BAX mediated mitochondrial outer membrane permeabili-zation (MOMP) and release of cytochrome c as well as apoptogenicproteins like apoptosis inducing factor (AIF) and endonuclease G(EndoG). Released cytochrome c contributes to a caspase-activatingcomplex that also contains apoptotic protease activating factor1 (Apaf1), and activates caspases resulting in caspase-dependentapoptosis, whereas AIF and EndoG can induce apoptosis indepen-dent of caspase activation. Ca2+ also activates calmodulin kinase II(CaMKII) and Ca2+ sensitive phosphatase calcineurin. Calcineurinhas been implicated to regulate activity of pro-apoptotic pro-tein Bcl2-associated agonist of cell death (BAD) and phosphatasesensitive nuclear factor of activated T-cells (NFAT) family transcrip-tion factors and activation of pro-apoptotic genes [81]. ActivatedIRE1 transmits apoptosis signaling through its kinase domain andrecruits the adaptor molecule TNF-receptor-associated factor 2(TRAF2). The ER stress induced IRE1–TRAF2 complex interacts withapoptosis-signal-regulating kinase (ASK1), leads to activation ofJNK, which suppresses anti-apoptotic activity of Bcl2 and promotespro-apoptotic activity of BIM via phosphorylation [81]. ER stress hasalso been shown to activate ER resident caspase12 by Ca2+ depend-ent calpain protease [81]. Activated caspase12 translocates fromthe ER to the cytosol where it cleaves caspase9, which induces thecleavage of the executioner caspase, caspase3, leading to apoptosisindependent of mitochondrial cytochrome c release.

Unlike higher eukaryotes, trypanosomatids lack IRE1, ATF6 aswell as CHOP and GADD34, which are involved in death responseduring ER stress. In T. brucei, following prolonged ER stress bytreatment with DTT or deoxy-glucose, the binding of the transcrip-tion factor tSNAP42 to promoters from which spliced leader (SL)RNA is transcribed is perturbed, thus inhibiting SL RNA production;this phenomenon is termed ‘SL RNA silencing’ (SLS) [12,13,82]. SLSeliminates trans-splicing of all mRNAs leading to a programmedcell death (PCD) pathway that is utilized by the parasites as apossible alternative to the apoptotic pathway observed in multi-cellular organisms. The regulating pathway that senses ER stressand spreads the signal to the nucleus, as well as the mechanismsunderlying the shut-off of SL RNA transcription awaits further char-acterization.

The successful induction of ER stress through the pharmaco-

logical inhibition of N-glycosylation by TM [71] is evidenced fromthe elevated expression of ER chaperone BiP. Protection from TMinduced cell death by chemical chaperone 4-phenyl butyric acid(4-PBA) further confirms the accumulation of misfolded proteins

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uring stress. It has been demonstrated that various stresses cannduce mitochondrial dysfunction, nuclease activation and DNAleavage in parasitic protozoa [83,84]. The molecular mechanismsf cell death in parasitic protozoa seem to be different with respecto multicellular organisms. Trypanosomatids lack key componentsf apoptotic pathway like death receptors, Bcl family proteinsnd caspases, so the classical caspases might be replaced in try-anosomes by other proteases. One possibility is the involvementf metacaspases (MCAs), which has been shown to execute apo-tosis in plants, fungi and protozoa [85]. Overexpression of theetacaspases in L. donovani (LdMCA1) or L. major (LmjMCA) pro-astigotes has been shown to make cells more susceptible to2O2-induced cell death through metacaspase-dependent mito-hondrial dysfunction [86,87]. However, analyses of metacaspaseene deletion mutants in L. mexicana (null for the MCA gene) [88],. brucei (a TbMCA2/TbMCA3/TbMCA5 null triple mutant) [89] andven in the unrelated apicomplexan parasite P. berghei (null forts MCA1 gene) [90] suggest that the encoded metacaspase generoducts are not responsible for regulated cell death. Thus theetacaspases of parasites cannot be appeared as caspase mimics

r, consequently, as mediators of a similar regulated cell death [91].ther peptidases (cathepsin L-like or cathepsin B-like families) ineishmania spp. are released from the lysosome during stress andontribute to the death of the parasite [92] but it is yet to be deter-ined whether lysosomal impairment designates a synchronized

r incidental event in cell death.

. Conclusions

In yeast and mammalian cells, ER stress initiates UPR branches inn intimately synchronized fashion for the homeostasis or apopto-is response. In contrast to higher eukaryotes, Leishmania possessesinimal components of the ER protein folding and quality con-

rol machinery and UPR stress/transducers. Previous publishedork on UPR stress/transducers of protozoan pathogens, including

eishmania, suggests the existence of a PERK translational controlathway and the lack of a UPR mediated transcriptional control.etailed studies are still required to understand the protein qual-

ty control and turnover under normal and stressed condition ineishmania parasites. In-depth studies are also required to exploreow the parasite sense the ER stress and transmit both adaptivend apoptosis signals. Critically, an apparently minimal UPR ineishmania and a susceptibility to ER stress, provide a novel tar-et for possible drug development that could possibly be extendedore broadly to other parasites, including the causative agents ofalaria, toxoplasmosis and trypanosomiasis.

cknowledgment

ER stress related work was supported by the National Bioscienceward for Career Development, DBT, India.

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