Caspase-Like Proteins: Acanthamoeba Castellanii Metacaspase and Dictyostelium Discoideum Paracaspase, What Are Their Functions?

Review

Caspase-like proteins: Acanthamoeba castellanii metacaspase and Dictyostelium discoideum paracaspase, what are their functions?

1,2 3 1, ENTSAR SAHEB , WENDY TRZYNA and JOHN BUSH * 1Biology Department, University of Arkansas at Little Rock, 2801 South University Ave., Little Rock, AR 72204-1099, USA 2Biology Department, University of Baghdad, Baghdad, Iraq 3Department of Biological Sciences, Marshall University, One John Marshall Drive, Huntington, WV 25755, USA

*Corresponding author (Fax, 501-569-3271; Email, [email protected])

Caspases are cysteine proteases that are important regulators of programmed cell death in animals. Two novel relatives to members of the caspase families metacaspases and paracaspase have been discovered. Metacaspase type-1 was identified in Acanthamoeba castellanii, an opportunistic protozoan parasite that causes severe diseases in humans. Paracaspase was found in the non-pathogenic protozoan Dictyostelium discoideum. Since their discovery in Acanthamoeba and Dictyostelium, metacaspases and paracaspases have remained poorly characterized. At present we do not have sufficient data about the molecular function of these caspase-like proteins or their role, if any, in programmed cell death. How these caspase proteins function at the molecular level is an important area of study that will provide insight into their potential for treatment therapies against Acanthamoeba infection and other similar parasitic protozoan. Additionally, finding the molecular functions of these caspase-like proteins will provide information concerning their role in more complex organisms.The aim of this article was to review recent discoveries about metacaspases and paracaspases as regulators of apoptotic and non-apoptotic processes.

[Saheb E, Trzyna W and Bush J 2014 Caspase-like proteins: Acanthamoeba castellanii metacaspase and Dictyostelium discoideum paracaspase, what are their functions? J. Biosci. 39 909–916] DOI 10.1007/s12038-014-9486-0

1. Introduction will provide further insight into their complete function in higher organisms. Additionally, findings pertaining to the Two novel relatives to the caspases have been discovered, molecular function of these proteins will contribute to the metacaspases and paracaspases (Uren et al. 2000). treatment therapies for parasitic protozoan infection. Metacaspases are found in many organisms such as fungi, plants, parasitic protozoa and some bacteria. On the other hand, paracaspases have been discovered in metazoans and 2. Types of cellular death in protozoan D. discoideum (Mottram et al. 2003; Carmona- Gutierrez et al. 2010). Several studies have presented differ- Several molecular pathways that lead to cell death have been ent views concerning the role of both metacaspases and identified as follows: necrosis, autophagy, mitotic catastro- paracaspases in programmed cell death. How these phe, program cell death and paraptosis (Sperandio et al. caspase-like proteins function at the molecular level remains 2000; Okada and Mak 2004). Cells that die in response to an important area of study. The cellular functional analysis accidental injury exhibit morphological changes – swelling of metacaspases and paracaspases in the amoebic protozoa and bursting – and release their internal contents into their

Keywords. Acanthamoeba; Dictyostelium; metacaspase; paracaspase http://www.ias.ac.in/jbiosci J. Biosci. 39(5), December 2014, 909–916, * Indian Academy of Sciences 909

Published online: 20 October 2014 910 Entsar Saheb et al. surroundings through the necrosis process. This event then contains the His/Cys catalytic dyad, a structure that is con- usually causes inflammation (Goodsell 2000). The second served in all caspases and necessary for substrate recognition type of cell death is autophagy. Eukaryotic cells respond to and catalysis (Aravind and Koonin 2002; Lamkanfi et al. certain external stress conditions (i.e. physical and chemical 2002). Two types of metacaspases have been distinguished: parameters) by undergoing rapid changes to protect them- type I metacaspases, which have a pro-domain region plus selves against potential damage (Kroemer et al. 2010). zinc finger motifs at the N-terminal, and type II Autophagic vacuoles have double membranes, metacaspases, which do not have a pro-domain; instead, they autophagosomes, that contain degenerating organelles and have approximately 180 amino acids inserted between the portions of the cytoplasm that are delivered to the lysosome p20 and p10 subunits. Recently, a novel group of (He and Klionsky 2009). Mitotic catastrophe is characterized metacaspases have been identified in phytoplanktonic pro- by the formation of multinucleate, giant cells. These cells tists, metacaspases type III. These metacaspases are distinct usually contain uncondensed chromosomes (Okada and Mak from type I and type II (Choi and Berges 2013). 2004). Cells also undergo controlled cell death known as Paracaspases have immunoglobulin (Ig) domain within a programmed cell death (PCD) (Goodsell 2000). PCD is a death pro-domain (Uren et al. 2000; Vercammen et al. genetically controlled mechanism that plays a crucial role 2004, 2007). Despite the similarity between caspases, in developing and maintaining health by eliminating aged metacaspases and paracaspases, caspases cleave their sub- or abnormal cells from organisms. Through the process of strates at the specific aspartate residue at the C-terminal side, apoptosis, there is a sequence of programmed events that while metacaspases have been shown to cleave their specific are regulated by a series of signals in the surrounding substrates at arginine/lysine residues or at the arginine area (Kerr et al. 1972; Arends and Wyllie 1991). (Vercammen et al. 2004, 2006; Watanabe and Lam 2005; Apoptotic cells shrink and are characterized by fragments Gonzalez et al. 2007; Tsiatsiani et al. 2011). Tudor of deoxyribonucleic acid (DNA) and other macromole- Staphylococcal Nuclease (TSN) is a conserved protein that cules that are packed into small vesicles and phagocy- is found in eukaryotes organisms (Yang et al. 2002). tosed by other cells (Bär 1996;Sperandioet al. 2000). In Decreasing the expression levels of TSN can trigger cell 2000, an alternative PCD has been named as paraptosis, death process (Tong et al. 1995). A study has revealed that which also requires gene expression. However, this kind metacaspase protein cleaves TSN at the Arg or Lys residues of PCD is characterized by cytoplasmic vacuolation and at the P1 position, while the cleave site for caspase-3 in the mitochondrial swelling without the presence of caspases human TSN is between the Tudor and SN5 domains in activity (Sperandio et al. 2000). apoptotic cells. Thus, TSN is a substrate for both metacaspases and caspases, and this is evidence that the cell death pathway is evolutionary conserved in plants and ani- 3. Molecular structure of caspases, metacaspases mals (Sundström et al. 2009). It has also reported that human and paracaspases paracaspase MALT1 cleaves after an arginine residue at the P1 position (Coornaert et al. 2008). Compared to caspases, Caspases are cysteine proteases (Cys-dependent Asp- metacaspases and paracaspases have differences in their specific peptidases) that are major regulators of PCD in structural features. Thus, they could be classified into a animals (Porter and Jänicke 1999). In addition, these en- distinct family of clan CD cysteine proteases (Vercammen zymes have also been shown to be involved in many non- et al. 2007). PCD processes such as inflammation, cell proliferation and cell differentiation (Lamkanfi et al. 2007). Caspases have been distinguished by the presence of a pro-domain at the N- 4. Role of metacaspases in PCD terminal region of a large subunit [such as the death domain (DD), caspase-recruitment domain (CARD) or death-effector Any PCD mechanism in unicellular organisms may serve as domain (DED)] followed by two catalytic domains (the large a protective mechanism that keeps their cellular populations p20 and small p10 subunit) (Lavrik et al. 2005; Timmer and best adapted for survival under stressful conditions, in addi- Salvesen 2007; Bouchier-Hayes et al. 2009). Caspases are tion to serving as a possible mechanism for managing cell expressed as pro-enzymes that are biologically inactive and cycles and differentiation/encystment (Koonin and Aravind require specific division of their large and small subunits, 2002). Theories have arisen from several studies reporting p20 and p10, respectively, in order to achieve maximum the involvement of metacaspases in PCD pathways. enzymatic activity (MacFarlane and Williams 2004; Functional studies in plants have proposed that metacaspase Launay et al. 2005; Fan et al. 2005). proteins may be associated with PCD. Arabidopsis Caspases and caspase-like proteins (metacaspases and metacaspases AtMC1 and AtMC2 were found to mediate paracaspases) belong to clan CD, family C14 cysteine pro- PCD in this plant (Coll et al. 2010). In yeast cells, it was teases. The active site of metacaspases and paracaspases shown that the Saccharomyces cerevisiae metacaspase Yca1

J. Biosci. 39(5), December 2014 Caspase-like proteins, Acanthamoeba, Dictyostelium 911 was involved in PCD of aged cells (Madeo et al. 2002). leading to palmitoylation of MCA4 protein (Proto et al. Yca1 is required to eliminate autophagic bodies that accu- 2011). Interestingly, another study reported that T. brucei mulate during yeast growth (Lee et al. 2010) and is involved metacaspases do not have a direct role in programmed cell in PCD when the yeast cells are subjected to different extra- death. Instead, those metacaspase proteins co-localized with cellular stresses such as viral infections and heat shock Rab11, which is a location indicator for recycling (Ivanovska and Hardwick 2005;Floweret al. 2005). endosomes. Thus, T. brucei metacaspases have functions Phytoplankton metacaspases and the metazoan caspase fam- other than PCD activity (Helms et al. 2006). Morever, a ily are orthologous, thus indicating that they may have group of proteases that lack a p10 domain was found in similar roles in cell death (Bidle and Falkowski 2004). several groups of eukaryotic phytoplankton. The same study In several protozoan parasites that infect humans, genes indicated that metacaspase type III has rearranged domain that express metacaspases have also been identified. In structures between the N- and C-terminii (Choi and Berges P. falciparum (malaria parasite), metacaspase1 (PfMCA1) 2013). Due to these facts, metacaspase proteins are not has been shown to induce apoptosis characterized by DNA always involved in PCD-directed apoptosis. fragmentation and disruption of transmembrane mitochon- Metacaspase genes are reported to be induced during drial potential (Meslin et al. 2007). When T. brucei various stress conditions. Arabidopsis thaliana metacaspase metacaspase 4 (TbMCA4) is expressed in S. cerevisiae, the gene (AtMCP2d) play a helpful role in reducing sensitivity transformed cells display growth reduction, respiratory dis- to oxidative stress inducers (Watanabe and Lam 2011). function and cellular death. In addition, this study reported Aspergillus fumigatus metacaspase genes, casA and casB, that metacaspases may control both nuclear functional pro- are not required for cell death; instead, these genes seem to cesses and the biogenesis of mitochondria (Szallies et al. play an important role in maintaining viability of the endo- 2002). T. cruzi, the causative agent of Chagas disease, has plasmic reticulum under stress conditions (Richie et al. two metacaspase genes, TcMCA3 and TcMCA5. Kosec et al. 2007). A study has shown that removing Candida albicans (2006) showed that these metacaspases may be involved in type I metacaspase, CaMCA1, could reduce oxidative stress apoptosis of the parasite cells. They demonstrated that both by de-stabilizing energy metabolism in this organism (Cao proteins (TcMCA3 and TcMCA5) change their subcellular et al. 2009). Schizosaccharomyces pombe (fission yeast) location when cells are subjected to fresh human serum metacaspase also have a role in growth and stress response (FHS)-induced PCD. In addition, when TcMCA5 was (Lim et al. 2007). In addition, it was determined that the over-expressed in T. Cruzi epimastigotes, the cells were LmjMCA-active catalytic domain has an important role in found to be more sensitive to FHS-induced PCD (Kosec disrupting mitochondrial functions in infected cells under et al. 2006). Regarding Leishmania parasite, it was reported oxidative stress or under anti-Leishmania drug-stressed con- that when L. major metacaspase (LmjMCA) is over- ditions (Zalila et al. 2011). expressed in S. cerevisiae yca1 null mutants and then sub- On the other hand, several studies indicate that jected to H O stress, PCD is induced. These results suggest 2 2 metacaspases play a role in growth regulation. MCA gene- that the LmjMCA protein is involved in the stress response deletion mutant in L. mexicana results in faster growth of the and functions similar to the endogenous stress response gene mutant amastigotes compared to wild-type amastigotes in YCA1 (Vercammen et al. 2006). macrophages cells; thus, L. mexicana MCA is a negative LdMCs are not responsible for the caspase-like activities regulator of the amastigote growth (Castanys-Muñoz et al. in L. donovani. However, upon exposure of the parasite cells 2012). Over-expression of P. falciparum PfMCA1 in yeast to the hydrogen peroxide stress, LdMCs play a role as effector molecules in Leishmania PCD (Lee et al. 2007). induced cellular growth inhibition (Meslin et al. 2011). Therefore, these studies strongly contribute to our understanding that metacaspases have functions other than PCD- 5. Role of metacaspases in non-PCD cellular functions related caspase activity such as cellular viability and/or stress signalling pathways. The function of proteins is determined by their domain Interestingly, it has been reported that metacaspases are structure (Marchler-Bauer et al. 2005). Even though the found in different stages of the life cycle, including the caspase-like proteins contain the structure that is conserved infectious stages. LmjMCA was found in different intracel- in all caspases, substitution of amino acids at critical residues lular compartments, such as the nucleus and kinetoplast, within the domain may alter the function of these proteins. during its parasitic cell cycle stages (Ambit et al. 2008). In To begin, T. brucei MCA4 metacaspase’s active site has a addition, it has been determined that LmjMCA is localized to serine residue instead of the normal cysteine (Szallies et al. the cytoplasm (Zalila et al. 2011). L. donovani LdMC1 and 2002; Mottram et al. 2003). It was shown that the T. brucei LdMC2 are expressed in the promastigotes and axenic MCA4 is in fact a pseudopeptidase that has no detectable amastigotes (Lee et al. 2007). T. brucei MCA2 and MCA3 peptidase activity and is exclusively processed by MCA3 genes are expressed in the mammalian bloodstream stage,

J. Biosci. 39(5), December 2014 912 Entsar Saheb et al. while MCA5 is expressed in the portion of the life cycle cell mitosis. This form can then become a cellulose-walled occuring in the insect vector (Helms et al. 2006). cyst via the encystation process, which occurs under Additionally, it was shown that T. brucei MCA4 is important unfavourable conditions (Moon et al. 2008; Leitsch et al. for parasite virulence during mammalian infection because it 2010). An initial sequence survey of approximately half of is expressed primarily in the bloodstream form of the para- the genome predicted that A. castellanii has a larger biosyn- site's life cycle (Proto et al. 2011). T. cruzi metacaspases thetic capability than either the parasite Entamoeba play an important role in the part of the parasite's life cycle histolytica or the slime mold D. discoideum. This explains occuring in the mammalian host. This metacaspase has a role the ability of A. castellanii to survive in various environ- in the parasite's differentiation of the infective stages ments (Anderson et al. 2005) including public water sup- (Laverrière et al. 2012) and is expressed in various stages plies, swimming pools, contact lenses, skin lesions and of the parasite's life cycle (Kosec et al. 2006). P. berghei cerebrospinal fluid (Khan 2003; Di Gregorio et al. 1992; metacaspase PbMC1 is expressed in female gametocytes and Schuster and Visvesvara 2004). Acanthamoeba causes sev- the mosquito life cycle stages but not in the blood eral diseases in humans (Zanetti et al. 1995). It can cause stages (Le Chat et al. 2007). granulomatous amoebic encephalitis, a central nervous system infection that occurs in patients with acquired immune deficiency syndrome and that almost always results in death 6. Cellular function of paracaspase, (Di Gregorio et al. 1992). It can also cause amoebic keratitis, another non-traditional caspase a painful disease of the cornea (Zanetti et al. 1995) that can cause damage to the eye tissue and lead to blindness Not much is known about the molecular function of (Marciano-Cabral and Cabral 2003). Human diseases caused paracaspase proteins. However, some studies have demon- by Acanthamoeba species still pose a problem because they strated some potential functional roles of paracaspase pro- are very difficult to treat. The cyst of the Acanthamoeba teins in humans. It has been demonstrated that paracaspase species life cycle has a rigid double-layered wall that pro- expressing mucosa-associated lymphoid tissue 1 (MALT1) tects the parasite against the host’s immune response, and in humans has an alternative caspase function. Diffused large many biocides have failed to kill the extremely resistant B cell lymphoma (DLBCL) is recognized by constitutive cysts. Surgery often proves unsuccessful and may result in NF-κB activity. It was shown that treating DLBCL cells numerous complications (Kumar and Lloyd 2002;Moon with the paracaspase MALT1 protease inhibitor blocks et al. 2008; Leitsch et al. 2010). A better understanding of A20 and BCL10 cleavage, the negative regulator of NF- the encystation process and the key proteins that participate κB. This, in turn, reduces the expression of NF-κB target in this process would aid in the search for more effective genes. Thus, paracaspase MALT1 protease inhibition could treatments for the diseases caused by this dangerous parasite. be a novel drug target for aggressive B cell lymphoma treatment (Ferch et al. 2009; Vucic and Dixit 2009). It has In addition to medical importance, Acanthamoeba is also also been hypothesized that MALT1 induces B cell antigen well known as a good model system to study eukaryotic cell receptor BCR to activate NF-κB of the adapter molecule B biology due to its relatively large size, rapid growth in cell CLL/lymphoma10 BCL10 (Dufner and Schamel 2011). culture and active motility (Moon et al. 2009). Moreover, it was shown that there is a functional relationship D. discoideum is a non-pathogenic unicellular eukaryotic between the caspase and paracaspase families in facilitating microorganism. The asexual life cycle of Dictyostelium in- non-apoptotic phenomena within these cells. The same study cludes two phases: the vegetative growth phase and a devel- also demonstrated that MALT1 activates caspase-8 during opment phase (Escalante and Vicente 2000). This amoeboid antigenic enhancement (Kawadler et al. 2008). Paracaspases protozoan is a powerful system to study gene function are known well in Dictyostelium (compared to through genetic and functional analysis. It has a haploid Acanthamoeba metacaspases) and it is generally appreciated genome of 34 Mb that contains many genes similar to higher that they need not be involved in Dictyostelium cell death eukaryotes and are absent in S. cerevisiae (Eichinger et al. (Roisin-Bouffay et al. 2004). 2005). In addition to having a relatively rapid life cycle, D. discoideum can grow at room temperature in a fairly inexpensive medium. Moreover, it is straightforward to track 7. Acanthamoeba and Dictyostelium are models Dictyostelium’s individual cells and the phenotypes of mu- for metacaspase and paracaspase analysis tants via developed genetic tools. These advantages make Dictyostelium a useful tool for examining cellular processes Acanthamoeba is a free-living soil amoebae and opportunis- that play essential roles in health and disease, and thus, these tic protozoan parasite. It has a two-stage life cycle: vegeta- organisms are being used to answer a wide range of biolog- tive trophozoite and an encysted form (Clark and Niederkorn ical questions (Raper 1935; Noegel and Schleicher 2000; 2006). Trophozoites undergo movement, phagocytosis and Eichinger et al. 2005; Gaudet et al. 2007;Aryaet al.

J. Biosci. 39(5), December 2014 Caspase-like proteins, Acanthamoeba, Dictyostelium 913

2008). The two model organisms Aanthamoeba and paracaspase has the Cys/His catalytic dyad, but does not Dictyostelium, each containing a non-traditional caspase, have the death domain and immunoglobulin domains are being used to gather insight into the novel roles that that are present in metazoan paracaspases (Uren et al. these enzymes may have in or outside of PCD. Especially 2000). In Dictyostelium, it has been reported that important would be results pointing to proteins or processes paracaspase-null mutant (pcp-) cells do not alter the that could be targeted for treatment of parasitic infections process of developmental stalk cell death and vacuolar (Table 1). autophagic cell death compared to wild-type cells (Roisin-Bouffay et al. 2004). A further study has shown that the autophagic and necrotic cell death processes are 8. A. castellanii metacaspase and D. discoideum not affected in the double mutants pcp- atg1- (Lam et al. paracaspase discovery and function 2007). The question remains that if they are not involved in vacuolar autophagic or necrotic cell death, Metacaspases and paracaspases have recently been identified what is their molecular function in the cell? in A. castellanii and D. discoideum, respectively (Uren et al. A. castellanii and the slime mold D. discoideum are 2000; Trzyna et al. 2008). The A. castellanii metacaspase closely related amoebozoa (Keeling et al. 2005). Both mi- contains the Cys/His catalytic dyad and a conserved region croorganisms have been applied as model organisms in with 30 amino acids that exists in all metacaspases. This different biological studies. A current study in metacaspase has a proline-rich N-terminal region. The initial Dictyostelium investigates the localization and the molecular cDNA sequence for metacaspase type-1 (NCBI, ID: function of the A. castellanii metacaspase (Acmcp) and AF480890) was obtained from a cDNA library sequenced D. discoideum paracaspase (Ddpcp) proteins. The study by Trzyna et al.(2008). Until recently, we did not have determined the intracellular location of both proteins via information about the localization or molecular function the creation of two cell lines of Dictyostelium that express of metacaspase protein in A. castellani. On the other the full length of Acmcp and the full length of Ddpcp, hand, a full-length sequence of the novel paracaspase respectively, using GFP-tagged protein. Furthermore, a com- gene was identified in D. discoideum using the PSI- parative functional analysis between Acmcp and Ddpcp was BLAST program (Uren et al. 2000). Paracaspases show conducted in the same study through mutagenesis and bio- different domain structures. Dictyostelium has a single chemical inhibition methods. The results of these studies aid paracaspase (Roisin-Bouffay et al. 2004). Dictyostelium in the verification of caspase-like protein localization and

Table 1. Metacaspases role in either PCD or cell viability/stress function

Organism Metacaspase name Metacaspase role References

A. thaliana AtMC1, AtMC2 PCD Coll et al. 2010 A. thaliana AtMCP2d Reducing sensitivity to oxidative stress inducers Watanabe and Lam 2011 A. fumigatus casA, casB Maintaining viability of the endoplasmic Richie et al. 2007 reticulum under stress conditions C. albicans CaMCA1 Reduce oxidative stress by de-stabilizing Cao et al. 2009 energy metabolism S. cerevisiae Yca1 PCD Madeo et al. 2002 S. pombe pca1(+) Growth and stress response Lim et al. 2007 P. falciparum PfMCA1 PCD Meslin et al. 2007 P. falciparum PfMCA1 Induced cellular growth Meslin et al. 2011 inhibition in yeast P. berghei PbMC1 Expressed in female gametocytes and the Le Chat et al. 2007 mosquito life cycle stages T. brucei TbMCA4 PCD Szallies et al. 2002 T. brucei TbMCA4 Parasite virulence during mammalian infection Proto et al. 2011 T. cruzi TcMCA3, TcMCA5 Parasite′s differentiation of the infective stages Laverrière et al. 2012 T. cruzi TcMCA3, TcMCA5 PCD Kosec et al. 2006 L. major LmjMCA PCD Vercammen et al. 2006 L. donovani LdMC1 and LdMC2 PCD Lee et al. 2007 L. mexicana LmxM A negative regulator of amastigote growth Castanys-Muñoz et al. 2012

J. Biosci. 39(5), December 2014 914 Entsar Saheb et al. function. The original observations concerning the eukary- References otic caspase-like proteins Acmcp and Ddpcp suggested that they were associated with both localization and the function- Ambit A, Fasel N, Coombs GH and Mottram JC 2008 An essential ing of the contractile vacuolar system (Saheb et al. 2013a, b). role for the Leishmania major metacaspase in cell cycle progres- The study suggests that Acmcp and Ddpcp in Dictyostelium sion. Cell Death Differ. 15 113–122 has a role in the formation, regulation and/or function of the Anderson IJ, Watkins RF, Samuelson J, Spencer DF, Majoros WH, contractile vacuol (an organelle critical for maintaining the Gray MW and Loftus BJ 2005 Gene Discovery in the normal morphology of the cell). The evidence suggests a role Acanthamoeba castellani Genome. Protist 156 203–214 in the plasma membrane fusion of contractile vacuoles, which Aravind L and Koonin EV 2002 Classification of the caspase- block water release. Moreover, a primary result has shown that hemoglobinase fold: detection of new families and implica- both proteins (Acmcp and Ddpcp) interact with other proteins tions for the origin of the eukaryotic separins. Proteins 46 – that are associated with the contractile vacuole in Dictyostelium 355 367 Arends MJ and Wyllie AH 1991 Apoptosis: mechanisms and roles (Saheb et al. 2013a, b). in pathology. Int. Rev. Exp. Pathol. 32 223–254 Unfavourable conditions, such as starvation, too high/low Arya R, Bhattacharya A and Saini SK 2008 Dictyostelium temperature and/or osmotic changes, cause cellular stress- discoideum – a promising expression system for the production related responses that can include caspase protein functions of eukaryotic proteins. FASEB J. 22 4055–4066 similar to the metacaspase-associated stress response func- Bär PR 1996 Apoptosis – the cell's silent exit. Life Sci. 59 369–378 tions reported in Schizosaccharomyces pombe and Bidle KD and Falkowski PG 2004 Cell death in planktonic, Aspergillus fumigatus (Cao et al. 2009; Watanabe and Lam photosynthetic microorganisms. Nat. Rev. Microbiol. 2 643– 2011). Processes involving the caspases-like proteins in uni- 655 cellular organisms that are capable of either encystment or Bouchier-Hayes L, Oberst A, McStay GP, Connell S, Tait SW, development to form stalk and spore cells seem to be a Dillon CP, Flanagan JM, Beere HM, et al. 2009 Characteriza- functional alternative to the caspase PCD pathways. The tion of cytoplasmic caspase-2 activation by induced proximity. Mol. Cell 35 830–840 finding that Acmcp and Ddpcp share the same location and Cao Y, Huang S, Dai B, Zhu Z, Lu H, Dong L, Cao Y, Wang Y, function within the cell provides new insight into the possi- et al. 2009 Candida albicans cells lacking CaMCA1-encoded bility that the caspase-like proteins in the protozoan amoeba metacaspase show resistance to oxidative stress-induced death may share similar functions. and change in energy metabolism. Fungal Genet. Biol. 46 183– 189 Carmona-Gutierrez D, Frohlich KU, Kroemer G and Madeo F 2010 9. Conclusions Metacaspases are caspases. Doubt no more. Cell Death Differ. 377–378 A new area of study has opened up to distinguish Castanys-Muñoz E, Brown E, Coombs GH and Mottram JC 2012 metacaspase and paracaspase cellular functions. The Leishmania mexicana metacaspase is a negative regulator of caspase-like activities of metacaspases and paracaspases in amastigote proliferation in mammalian cells. Cell Death Dis. 3 cell death and other cellular functions (stress-related) have e385 been studied in many organisms. Although metacaspases Choi CJ and Berges JA 2013 New types of metacaspases in phy- and paracaspases are caspase-like proteins, they may or toplankton reveal diverse origins of cell death proteases. Cell may not be involved in PCD. The molecular function of Death Dis. 4 ARTN e490. doi 10.1038/cddis.2013.21 A. castellanii metacaspase and D. discoideum paracaspase Clark DW and Niederkorn JY 2006 The pathophysiology of – are not yet well understood and require further study. Acanthamoeba keratitis. Trends Parasitol. 22 175 180 Coll NS, Vercammen D, Smidler A, Clover C, Van Breusegem F, However, findings concerning the location of A. castellanii Dangl JL and Epple P 2010 Arabidopsis type I metacaspases metacaspase within cells provide information about their control cell death. Science 330 1393–1397 potential as drug targets for future treatment. Confirmation Coornaert B, Baens M, Heyninck K, Bekaert T, Haegman M, Staal the role of paracaspases and metacaspases in cellular stress- J, Sun L, Chen ZJ, et al. 2008 T cell antigen receptor stimulation response pathways in Dictyostelium development and/or induces MALT1 paracaspase–mediated cleavage of the NF-κB Acanthamoeba encystation will provide an understanding inhibitor A20. Nat. Immunol. 9 263–271 of how these two essential processes occur and how they Di Gregorio C, Rivasi F, Mongiardo N, De Rienzo B, Wallace S may be prevented during parasitic protozoan infections. and Visvesvara GS 1992 Acanthamoeba meningoencephalitis in a patient with acquired immunodeficiency syndrome. Arch. Pathol. Lab. Med. 116 1363–1365 Acknowledgements Dufner A and Schamel WW 2011 B cell antigen receptor- induced activation of an RAK4-dependent signaling path- We greatly appreciate Dr Marinelle Ringer for her assistance way revealed by a MALT1-IRAK4 double knockout mouse in proofreading the manuscript. model. Cell Commun. Signal. 9 6

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MS received 03 February 2014; accepted 29 September 2014

Corresponding editor: SUDHA BHATTACHARYA

J. Biosci. 39(5), December 2014