Types of cyclophilins

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2.1 The peptide bond:

Peptide bond is a covalent bond, made from condensation reaction of carboxyl group of amino terminal amino acid and amino group of carboxy terminal amino acid by releasing a water molecule. This, reaction appears during event of translation which is a basic event for formation of variety of proteins for attaining various cellular functions. Further, peptide bond is a partial double bond in nature that makes the amide group in planar conformation and hinders the rotation of amide group through the amide bond (Ramachandran and Sasisekharan, 1968). Because of this restriction in bond rotation, peptide bonds usually exist in trans from, in which side chains of both the adjacent amino acids are in 180⁰. Due to which the peptide bonds which are in trans form are more thermodynamically and sterically stable and favored compared to cis form peptide bonds.

However, peptide bonds preceeding a proline amino acid, the scenario is different. Because of the peptidyl prolyl imide bond in proline, the difference in free energy between cis and trans isomers is smaller compared to other amino acids (Lu et al., 2007). In total, cis conformation occurs in 6.5% of proline containing peptide bonds (X-pro peptide bond) but for the peptide bond without proline, it is only 0.05% (Stewart et al., 1990). This is because the proline amino acid has cyclic side chain with no alpha hydrogen to make hydrogen-bonds in alpha helices and beta sheets, which makes it unsuitable for alpha helix region. In contrast, cis rotamer fits well in turn, bend, and coil region during protein folding. The isomerizations about proline imidic bonds are regarded as the rate-determining events in protein folding. This inter conversion of cis/trans isomerization in peptidyl prolyl bond is performed by an enzyme called peptidyl prolyl cis/trans isomerase (PPIase).

2.2 Peptidyl-prolyl cis-trans isomerases (PPIase)

Petidyl-prolyl cis-trans isomerases (EC are the group of enzymes which assists in protein folding. Unlike other chaperon proteins which also assist in protein folding, PPIases are involved in interconversion of cis and trans isomers of the peptidyl-prolyl (Xaa-Pro) bonds in peptide and protein and facilitates protein folding in vivo and in vitro (Gething and Sambrook, 1992). PPIase assists in protein folding by isomerization of peptidyl prolyl bond. Compared to other protein folding steps, isomerization of peptidyl prolyl bond is considered as the rate limiting step in protein folding (Brandts et al., 1975). PPIase activity is especially important during stress conditions where a change in physiological condition denatures the correctly folded protein. It is essential for the protein to be in correct three-dimensional state for its function, PPIases are involved in the salvage of such misfolded proteins during the stress conditions. These PPIases were isolated for the first time from porcine kidney cortex by Fisher et al. (1984). There are four classes of enzymes comes under PPIases. (1) parvulins (2) FK506 binding proteins (FKBP) (3) cyclophilins (CYPs) (4) phosphatase 2A phosphatase active

ator (Jordens et al., 2006). All these class of proteins are having Peptidyl prolyl cis-trans isomerization activity. FKBP’s and CYP’s together termed as immunophilins as they both are targets of immune suppressive drugs FK506 and cyclosporine respectively. Parvulins a small proteins having only 92 amino acids have PPIase activity, first discovered and isolated from E.coli by ; Rahfeld et al., 1994. Besides PPiase activity this parvulins also have choperonin activity. Cyclophilin A was first identified and purified while screening for the intracellular protein target of cyclosporin A (CsA), a fungal metabolite with potent immunosuppressive activity (Handschumacher et al., 1984). All classes of PPIases are ubiquitous in nature present in bacteria, fungi, yeast, higher plants including humans which suggest that they are involved in crucial role.

2.3 Cyclophilins:

Cyclophilins are ubiquitous in nature present in bacteria to higher plants and animals. They are also called as immunophilins, as they are targets of immune suppressive drugs like cyclosporineA. As already explained, cyclophilins are involved in rotation of amino terminal peptide bond of proline from cis to trans confirmation. During protein biosynthesis most of the peptide bonds are in trans confirmation, which is a low free energy state and thermodynamically stable. However, due to cyclic structure of proline the peptide bonds preceeding to proline more often found in cis conformation (Stewart et al., 1990; Weiss et al., 1998). Due to the resonance stabilizing effect of the imide bond, the rotation of Xaa-Pro peptide bonds was proposed to be the rate limiting step in protein folding (Brandts et al., 1975) and the reaction catalyzed by cyclophilins is important in improving folding rates. Peptidy prolyl cis - trans isomerase activity has been shown to accelerate refolding of several proteins in vivo (Fischer et al., 1984; Kern et al., 1995). Various cyclophilins were used to accelerate the refolding process of Rnase T1 which appeared to be most efficiently catalyzed by CyP-18 (Fischer et al., 1985). PPIases along with bovine serem albumin (BSA) aids in the process of correct in vitro refolding of antibodies (Lilie et al., 1993). In vivo roles for CyP in protein folding have been demonstrated (Lodish et al., 1991; Matouschek et al., 1995; Rassow et al., 1995; Smith et al., 1995; Steinmann et al., 1991).

Types of cyclophilins:

Human cyclophilins:

The human genome encodes 16 unique CYPs which are categorized into 7 major groups viz; human CYP A (hCYP-A), hCYP-B, hCYP-C, hCYP-D, hCYP-E, hCYP-40 and hCYP-NK (Galat, 2003; Waldmeier et al., 2003). The hCYP-A binds CsA, and forms a ternary complex with calceneurin. CsA-hCYP-A binding to calceneurin inhibits the phosphatase activity of calceneurin. As a result, the transcription factor, nuclear factor of activated T cells (NFAT), remains inactive in the cytoplasm and the interleukin-2 gene is not transcribed, leading to the inactivation of T-cells (Liu et al., 1991b). Extensive studies have been carried out with hCYP-A, the first CYP to be identified. hCYP-A plays a crucial role in the protein folding process as it possesses both catalytic and chaperone-like activities. For example, hCYP-A promotes the formation and infectivity of human immunodeficiency virus (HIV-1) virions (Braaten and Luban, 2001). hCYP-A is incorporated into HIV-1 virions where it interacts with the HIV-1 Gag protein, a polyprotein precursor of virion structural protein. The presence of four conserved proline residues in HIV-1 is crucial for incorporation of hCYP-A into virions (Franke et al., 1994; Thali et al., 1994). Similarly, other human CYPs are found to be associated with different signal transduction pathways, cell signaling pathways, regulation of gene expression, and immune response (reviewed in Lu et al., 2007). The hCYP-A consists of eight stranded anti-parallel β-barrel with two α-helices surrounding the barrel (Kallen et al., 1991). Study using site directed mutagenesis of W 121 A (replacing tryptophan-121 with alanine) did not inhibit phosphatase activity of calceneurin, suggesting its critical role in CsA binding, with no change in PPIase activity (Liu et al., 1991a; Zydowsky et al., 1992). In contrast, mutation in Arg-55, Phe-60 and His-126 inhibited PPIase activity by 99% (Zydowsky et al., 1992). The exact mechanism of bond rotation by CYP during catalysis is not yet known. However, it is believed that the conserved arginine in the hydrophobic active site of hCYP-A makes a hydrogen bond with the peptide nitrogen, resulting in a peptidyl-prolyl bond with more single-bond character.

Plant CYPs

Very little is known regarding plant CYPs compared to human CYPs. The first plant CYPs were identified concurrently from tomato (Lycopersicon esculentum), maize (Zea mays), and oilseed rape (Brassica napus) (Gasser et al., 1990). Then after, with the availability of whole genome sequencing, the identification and characterization of plant CYPs has progressed significantly. So far, 62 number of CYPs were reported in Glycin max which is a highest number reported in plant species (Mainali et al., 2014). Apart from Glycin max, Arabidopsis thaliana and Oryza sativa are the two other plant species reported with 31 AtCYPs (Kumari et al 2014) and 28 OsCYPs (Ahn et al., 2010; Trivedi et al., 2012) respectively. However, compared to other organisms, the total number of plant CYPs in databases is still small, which suggests that many plant CYPs remain to be identified (Opiyo and Moriyama, 2009).

A total of 62 CYPs were reported in soybean (Mainali et al., 2014). Out of which, 51 are having single domain i.e cyclophilin like domain (CLD) and remaining 10 are having multi domains. GmCYP8, GmCYP9, GmCYP16 and GmCYP17 are having two tetratricopeptide repeats (TPR) at C-terminal region of catalytic domain (CLD). GmCYP20 and GmCYP35 have tryptophan-Aspertate repeat (WD) at N-terminal region, GmCYP56 and GmCYP59 contain RNA recognition motif (RRM) and Zink Knuckle (ZK) domains at C-terminal region and GmCYP18 and GmCYP19 have U-box domain at N-terminal region. Among the 62 GmCYPs, it was ascertained that 13 contain a chloroplast transit peptide, 13 contain a signal peptide, 5 contain a mitochondrial targeting peptide, 10 contain a nuclear localization signal, and the remaining 21 are cytosolic. Unlike Arabidopsis and rice CYPs none of the soybean CYPs are predicted to be localized to the ER or golgi or plasma membrane. However, GmCYP39 is predicted for localization in the mitochondrial inner membrane or plasma membrane. All the 62 GmCYPs are distributed in 18 chromosomes; No CYPs are present in chromosome 8 and 16. Most of the GmCYP genes showed distinct tissue-specific expression pattern. Out of the 62 GmCYP genes, 26 were expressed in the vegetative tissues whereas, 34 were expressed in floral buds and different stages of seed development (Mainali et al., 2014).

A total of 31 CYPs were reported in Arabidopsis thaliana (Kumari et al., 2014). Among the identified AtCYPs, only some were characterized at the molecular level. Out of 31, 14 were characterized (Romano et al., 2004; Gullerova et al., 2006; Li and Luan, 2011). Among them, five are cytoplasmic cyclophilins (Hayman and Miernyk,1994; Chou and Gasser, 1997), two are endoplasmic reticulum (ER) localized isoforms (Jackson and Soll, 1999; Grebe et al., 2000), six are chloroplast isoforms (Lippuner et al., 1994; Schubert et al., 2002; Peltier et al., 2002), and one is nucleus localized cyclophilin (Gullerova et al., 2006). Out of 31 cyclophilins, 21 are single domain (SD) containing genes i.e cyclophilin like domain (CLD) and the remaining 10 are multi domain cyclophilins. The SD cyclophilins are further distinguished by the absence (AtCYP18-1, AtCYP18-2, AtCYP18-3, AtCYP18-4, AtCYP19-1, AtCYP19-2 and AtCYP19-3) or presence (AtCYP19-4, AtCYP20-1, AtCYP20-2, AtCYP20-3, AtCYP21-1, AtCYP21-2, AtCYP21-3, AtCYP21-4, AtCYP23-1, AtCYP28, AtCYP26-2 and AtCYP37) of an N-terminal targeting sequence. However, with in the SD cyclophilins, AtCYP23-1 and AtCYP26-1 have unique pre-dicted transmembrane domains located at the N- andC-termini, respectively. The multi domain cyclophilins have additional domains along with CLD. AtCYP71 contains WD40 repeat and AtCYP65 has U repeat at N terminal side. Whereas, AtCYP57 has Ser/Lys-Arg/Glu-rich region (S/K, R/E) and AtCYP40 contain Tetratrico peptide repeat (TPR) domain at C-terminal side of cyclophilins. AtCYP59 contains both RNA recognition motif (RRM) and Arg-rich region at C terminal side, AtCYP 63 and 95 contains Arg-Ser rich regions (RS) at C terminal end of cyclophilin and both these isoforms are also characterized by the presence of a nuclear localization signal adjacent to the cyclophilin domain (Romano et al., 2004). Presence of RNA-recognition domains in MD cyclophilins indicates that they participate at different stages within the mRNA processing pathway.

Rice is considered as a model crop plant. The completion of the rice genome sequencing project in 2005 (International Rice Genome Sequencing Project, 2005), and the availability of the sequences in the public domain has facilitated the ability of researchers to carry out genome wide analysis of the gene families. However, compared to the Arabidopsis CYPs, little work has been done on the rice CYPs. Genome wide analysis of the rice genome revealed 29 CYPs genes (Kumari et al., 2014) encoding 46 putative proteins instead of 27 (Ahn et al. 2010) and 28 (Trivedi et al. 2012) gene members reported previously. Further, 18 OsCYPs are single domain proteins having CLD as catalytic domain and the remaining are Multi domain proteins having an additional domain along with CLD. They also contain more divergent functional domains, such as the TPR domain, the WD-40 repeat, the U-box domain and the Zinc finger, each of which is involved in protein-protein or protein-DNA interactions. An RNA recognition motif (RRM) that may interact with RNA was also found. 6 of the 9 MD CYPs have Arg/Lys amino acid-rich domains that may function as a motif for non-specific RNA-binding or mediate protein- protein interactions, and are also frequent targets of molecular interactions (Ahn et al., 2010). Interestingly, Cyclophilin genes with multiple CLDs have not been found so far in mammals as well as in plants but have been reported in lower prokaryotes (Barik 2006).

Plant CYPs are involved in stress response

Because plants are sessile in nature, plants have to be able to endure a wide range of biotic and abiotic stresses in entire their life cycle. Abiotic stresses arise due to one or combined effect of water deficit, water logging, high salinity, extreme temperature, high radiation, chilling injury, heavy metal toxicity and pH. Biotic stresses are due to the attack by pathogenic organisms like viruses, bacteria, fungi, and nematodes. In order to combat these stresses, plants cope up different mechanisms at the cellular and molecular level (Fujita et al., 2006; Atkinson and Urwin, 2012). All plants can sense and transmit stress stimuli signals to trigger different cell signaling cascades involved in ion channels regulation, kinase function, hormones like salicylic acid, ethylene, jasmonic acid, and abscisic acid action and pathways related reactive oxygen species (ROS) scavenging. These signaling events altogether induce expression of defense genes that finally lead to the entire defense reaction response. However, in all stresses a common phenomenon has been happened i.e denaturation of cellular proteins which can result in cell death. To tackle this situation, special molecules called choperons stabilize/renatures protein molecules inside the cell and protect the cell from dying. The chaperone-like activity of CYPs and their role in the rate limiting step of protein folding by peptidyl prolyl bond isomerization (Brandts et al., 1975) is associated with their involvement in different types of stress responses. Expression of many plant CYPs is induced in response to several different types of abiotic stresses suggesting their possible function in stress tolerance.

Many reports have been published in related to plant cyclophilins and their expression in stress responses. Interestingly, cyclophilin expression has reported not only under abiotic stress conditions but also under biotic stresses including HgCl2 ( Marivet et al., 1992), viral infection, salicylic acid, salt stress, heat and cold shock (Marivet et al., 1994, 1995;Scholze et al., 1999; Godoy et al., 2000), light (Chou and Gasser, 1997; Luan et al., 1994), drought (Sharma and Singh, 2003), wounding, fungal infection, abscisic acid, and methyl jasmonate (Godoy et al., 2000; Kong et al., 2001). The halophyte Thellugiella halophila cyclophilin expression is highly inducible by salt, abscisic acid (ABA), H2O2 and heat shock (Chen et al., 2007).

For example, expression of the Arabidopsis CYP, ROTAMASE CYCLOPHILIN 1 (ROC1), increases upon wounding (Chou and Gasser, 1997).

Similarly, transcription of maize and bean CYPs increase under conditions of heat-shock, wounding, high salinity, or low temperature (Marivet et al., 1992).

Moreover, Solanum commersonii CYP gene expression is up-regulated by low temperature, abscisic acid, drought, or wounding (Meza-Zepeda et al., 1998).

Pepper CYPs are differentially regulated during abiotic stress or pathogen infection (Kong et al., 2001).

Support for their role in stress response has also come from overexpression studies. Expression of Thellungiella halophile CYP, ThCYP1, is induced by salt, heat, abscisic acid, and hydrogen peroxide and ectopic expression of ThCYP1 in fission yeast and tobacco cells increases the tolerance to salt stress (Chen et al., 2007). Transgenic Arabidopsis expressing pigeon-pea CYP (CcCYP1) increases tolerance against drought, salinity and high temperatures, with enhanced PPIase activity under stressed conditions (Sekhar et al., 2010). Similarly, overexpression of cotton CYP (GhCYP1) in transgenic tobacco plants confers tolerance against salt stress and fire-blight disease (Zhu et al., 2011).

However, recent studies on rice CYPs show their roles in different types of stresses. OsCYP2 has been reported to have role in different abiotic stress responses (Ruan et al., 2011). The expression of OsCYP2 is up-regulated during salt stress, and its over-expression in rice enhances tolerance towards the salt stress. Similarly, overexpression of the thylakoid-localized OsCYP20-2 in Arabidopsis and tobacco provides increased tolerance towards osmotic stress, and to extremely high light condition (Kim et al., 2012). Moreover, there are several other OsCYPs found to be up-regulated by abiotic stresses like desiccation and salt stress (Ahn et al., 2010; Trivedi et al., 2012), indicating a critical role of OsCYPs during stress conditions.

Increase in the expression of ROC1 in response to light is found to be associated with phytochromes and cryptochromes (Chou and Gasser, 1997; Trupkin et al., 2012). roc1 mutants with a T-DNA insertion in the promoter region display an early flowering phenotype under long day, but not under short day photoperiod (Trupkin et al., 2012). However, loss-of-function mutants of AtCYP40 reduce the number of juvenile leaves, with no change in inflorescence morphology and flowering time (Berardini et al., 2001). Similarly, gain-of-function mutations in ROC1 reduce stem elongation and increase shoot branching (Ma et al., 2013). Moreover, AtCYP59, a multi-domain CYP with a RNA recognition motif (RRM), regulates transcription and pre-mRNA processing by binding to the C-terminal domain of RNA polymerase II (Gullerova et al., 2006). The Arabidopsis plants with a defective AtCYP20-3 are found to be hypersensitive to oxidative stress conditions created by high light levels, high salt levels, and osmotic shock (Dominguez-Solis et al., 2008). Collectively, these results show the roles of Arabidopsis CYPs in different cellular pathways, which necessitate further work to explore the functionality associated with each of the CYPs.

Sequence alignment of GmCYP1 with cotton CYP, GhCYP1, and pigeon pea CYP, CcCYP1, the expression of which are associated with stress response, shows 91% and 73% identity at amino acid level. Therefore I hypothesize that the GmCYP1 is associated with stress response in soybean.