Chapter Biological background CHAPTER 2. BIOLOGICAL BACKGROUND 2.1 DISCOVERY Transplantation of organs has been one of the most impressive advances in modern medicine. Surgeons are quite skillful at these demanding operations. However, all their skill would be a waste without the immunosuppressant drugs that are critical if patients are to survive. The main function of the immune system is to distinguish between self from non-self and to protect us from microbial invaders. At the same time, implanted foreign organs will be attacked and destroyed by our immune system. Until the discovery of the cyclosporins that suppress the immune system effectively to allow the acceptance of foreign organs, whole organ transplants (except in identical twins) were rarely successful in the long term. The cyclosporins (produced by several species of fungi) were discovered in the 1970’s, and by the 1980’s, the survival rates of those who received organ transplants rose dramatically. In 1984, Gunther Fischer, a German biochemist studying protein folding, described an 18 kDa protein isolated from the porcine kidney which catalyzed the interconversion of cis and trans rotamers of amide bonds adjacent to proline residues in peptidic substrates (Fig. 2-1). Fischer termed this enzymatic activity peptidylprolyl cistrans isomerase activity, and the enzyme became known as PPIase [Fischer, 1984]. 29 Chapter Biological background Figure 2-1. Cis-trans isomerization about peptidylprolyl bonds catalyzed by rotamases (immunophilins). In that same year, Handschumacher and colleagues investigating the cellular actions of the immunosuppressant drug cyclosporin A (CsA, Fig. 2-2) isolated a protein from the calf thymus that was the principal binding protein for CsA. They dubbed this protein cyclophilin (CyP) [Handschumacher,1984]. Figure 2-2. Immunosuppressant drugs which are selective inhibitors of the rotamase activity of the immunophilin FKBP12 (FK506 and rapamycin) or cyclophilin A (cyclosporin A). 30 Chapter Biological background By 1989, it became clear that PPIase and cyclophilin were the same protein [Fischer, 1989; Takahashi, 1989]. Around the same time, another immunosuppressant drug, the antibiotic FK506 (Fig. 2-2) was characterized and its target protein was identified [Harding, 1989; Siekerka, 1989]. This protein also bound tightly to the structurally related immunosuppressant drug rapamycin (RAPA). Although this new 12 kDa protein, called FKBP (for FK506 binding protein), had no sequence homology to cyclophilin, it too was shown to possess PPIase activity. All three drugs bound to the proline-binding site of their respective PPIase partners and potently inhibited their enzymatic activity. As it turns out, inhibition of PPIase activity is irrelevant for the immunosuppressive actions of CsA, FK506, and RAPA. It is the complex of the drugs with their cognate immunophilin in each case is the active immunosuppressant agent. 2.2 IMMUNOPHILINS AND IMMUNOSUPPRESSANT DRUGS The mechanisms of immunophilin-mediated immunosuppression elicited by FK506, RAPA, and CsA have been extensively studied and reviewed [Schreiber, 1991; Jin, 1992; Braun, 1995; Rosen, 1992; Armistead, 1993]. Although FK506 and CsA are structurally dissimilar (Fig. 2-2) while FK506 and RAPA are structural analogues of each other, FK506 and CsA share similar pharmacology, which is distinct from that of RAPA. Both FK506 and CsA inhibit Ca2+-dependent signaling pathways in T-cells emanating from activated T-cell receptors and resulting in transcription of genes for interleukin-2 (IL-2) and its receptor. The discovery that both immunosuppressants bound to proteins, 31 Chapter Biological background although very different in sequence and structure that possessed the same enzymatic activity suggested that inhibition of rotamase activity was the pharmacologically relevant activity. However, RAPA, which like FK506 binds to FKBP12 and potently inhibits its rotamase activity, acts at a later Ca2+-independent stage in T-cell signaling, blocking IL2-dependent entry of lymphocytes into the cell cycle and hence inhibiting cell proliferation [Bierer, 1990]. These two mechanistic pathways are distinct from each other, and FK506 and RAPA are reciprocal antagonists of each other's pharmacological effects [Dumont, 1990]. 2.2.1 Cyclosporin Cyclosporin A (CsA) is a cyclopeptide of fungal origin and Bueding et al first discovered its schistosomicidal activity in 1981. CsA is also active against other parasites from different phyla, including apicomplexa, nematodes and plathyhelminths. Cyclosporin A, a lipophilic undecapeptide, and FK506 (tacrolimus), a macrolide antibiotic, which are used clinically as potent immunosuppressor agents [Borel et al., 1996], have many effects in the nervous system such as modulation of the release of certain neurotransmitters, neurotrophic influences and protection against glutamateinduced neurotoxicity [Steiner et al., 1996; Kikuchi et al., 1998; Ruiz et al., 2000 ; Lyons et al., 1994]. These agents reduce the catalytic activity of neuronal nitric oxide synthase (nNOS) and subsequently cause the inhibition of NO release [Dawson et al., 1993; Sharkey and Butcher, 1994; Rao et al., 1996; Snyder et al., 1998]. This latter mechanism 32 Chapter Biological background has been implicated in some of the important functions of cyclosporin A in the nervous system [Sabatini et al., 1997; Ruiz et al., 2000; Sanchez-Lozada et al., 2000]. 2.2.2 FK506 FK506 is a new FDA-approved immunosuppressant used for the prevention of allograft rejection in, for example, liver and kidney transplantations. FK506 is inactive by itself and requires binding to FK506 binding protein 12 (FKBP-12), or immunophilin, for activation. In this regard, FK506 is analogous to cyclosporin A, which must bind to its immunophilin (cyclophilin A) to display activity. This FK506-FKBP complex inhibits the activity of serine/threonine protein phosphatase 2B (calcineurin), the basis for the immunosuppressant action of FK506. 2.2.2.1 Neurotrophic properties of FK506 The therapeutic relevance of the neurotrophic properties of FK506 was demonstrated by Gold et al., who showed that in rats with lesions of the sciatic nerve, treatment with FK506 enhanced both nerve regrowth and the regain of neurological function [Gold et al., 1994 & 1995]. The nerve regenerative property of this class of agents is separate from their immunosuppressant action because the FK506-related compounds that bind to FKBP-12 but not inhibit calcineurin are also able to increase nerve regeneration. Thus, FK506's ability to increase nerve regeneration arises via a calcineurin-independent mechanism (i.e., one not involving an increase in GAP-43 phosphorylation). Possible mechanisms of action are discussed in relation to known actions of FKBPs: the interaction of FKBP-12 with two Ca2+ release-channels (the 33 Chapter Biological background ryanodine and inositol 1,4,5-triphosphate receptors), which is disrupted by FK506, thereby increasing Ca2+ flux; the type receptor for the transforming growth factor β (TGF-β 1), which stimulates nerve growth factor (NGF) synthesis by glial cells. 2.2.3 Rapamycin Rapamycin (RAP), a lipophilic macrolide, was identified more than twenty years ago during antibiotic screening at Ayerst Research Laboratories. Produced by a strain of Streptomyces hygroscopicus isolated from a soil sample obtained from the Vai Atore region of Easter Island (Rapa Nui) [Vezina,1975], RAP is a white crystalline solid (m.p. 183-185 ºC), virtually insoluble in water but readily soluble in ethanol, methanol, dimethylsulfoxide, and other organic solvents [Sehgal,1975]. Although lacking antibacterial activity, RAP is a potent inhibitor of yeast growth and a moderate growth inhibitor of filamentous fungi [Sehgal, 1975]. It is most active against the species of Candida, particularly C. albicans, and protects against systemic and vaginal candidosis in mice, without acute toxicity. The first demonstration of RAP's immunosuppressive activity was obtained from studies showing its inhibitory effects upon production of humoral IgE as well as its preventative effects in two animal models of human autoimmune diseases, experimental autoimmune encephalitis and adjuvant arthritis [Martel, 1977]. 2.3 TARGETS FOR IMMUNOSUPPRESSION To understand the molecular mechanisms of immunosuppression by CsA, FK506, and rapamycin, the cellular receptors of these drugs have been purified and characterized 34 Chapter Biological background [reviewed by Schreiber, 1991; Fruman et al., 1994]. CsA binds to a family of receptors named cyclophilins (CyPs), and FK506 and rapamycin bind to a distinct set of receptors called FKBPs (standing for FK506 and rapamycin-Binding Proteins). Cyclophilins and FKBPs are collectively referred to as immunophilins [Schreiber, 1991].Interestingly, these receptor proteins have subsequently been shown to have peptidylproline cis-trans isomerase (PPIase or rotamase) activity [Harding et al., 1989; Fischer et al., 1989]. Kinetic and structural studies have shown that the enzyme activity of both CyPs and FKBPs are competitively inhibited by binding of their cognate ligands [Van Duyne et al., 1991]. Therefore, it was speculated that immunosuppression by these drugs is due to the loss of rotamase activity. Remarkably, despite their structural diversity both CsA and FK506 mediate their biological effects through inhibition of the same protein phosphatase calcineurin (CN) and inhibition of PPIase activity was found to be ancillary to the immunosuupressant effects. Recently, this argument has taken a new twist with the proposal that immunophilin-drug complexes block additional signal transduction pathways in activated T-cells and that these T-cell specific pathways may be significant contributors to the source of the T-cell specific effects of these compounds [Matsuda and Koyasu, 2003] 2.4 FUNCTIONS OF IMMUNOPHILINS Several lines of evidence suggest that low Mr immunophilins such as FKBP12 and cyclophilins A and B in complex with immunosuppressant drugs inhibit the activity of calcineurin, a Ca2+/calmodulin-dependent protein phosphatase, thereby blocking the 35 Chapter Biological background signaling pathway for T-cell activation [Liu et al., 1991; Clipstone and Crabtree, 1992]. High Mr immunophilins such as FKBP51, FKBP52, and Cyp40 have been identified as components of steroid-receptor complexes. Although it remains to be demonstrated that these immunophilins affect receptor action or heterocomplex assembly in any way [Hutchison et al., 1993; Dittmar et al., 1996], there is some evidence to suggest that FKBP52 may play a role in trafficking the receptor to the nucleus [Czar et al., 1995; Owens-Grillo et al., 1996a]. The high Mr immunophilins possess a TPR domain and a calmodulin-binding domain in their C-terminal half [Callebaut et al., 1992]. The TPR domains consist of 34 amino acid repeats with a degenerate consensus and are believed to be sites for protein-protein interactions [Sikorski et al., 1990]. The immunophilins in steroid-receptor complexes bind to a common site on Hsp90 via their TPR domains [Radanyi et al., 1994; Owens-Grillo et al., 1995]. Many mammalian FKBPs are widely expressed in the brain, and a role in nervous system development has been suggested. FK506 was found to have neurotrophic effects in PC12 cells and sensory ganglia, as well as stimulating regrowth of crushed sciatic nerves in vivo. 2.4.1 Cellular functions of immunophilins Rates of folding of proteins vary considerably, and fast- and slow-folding forms of unfolded proteins have been detected. Isomerization about peptidylprolyl amide bonds is one of the slower steps in protein folding and thus may represent a rate-limiting step in 36 Chapter Biological background protein folding and unfolding [Schmid, 1993]. CyPA has been shown to accelerate the in vitro refolding of several proteins, including immunoglobulin chains [Lang, 1987; Lilie, 1995], carbonic anhydrase [Freskgard, 1992] and Rnase T1. Human bradykinin is likewise a substrate for CyPA [London, 1995] as is calcitonin [Kern, 1993]. The ability of CyPA to catalyze protein refolding is inhibited by CsA. FKBP12 has also been shown to catalyze the folding of carbonic anhydrase and RNase T1, with less efficiency than CyPA. CyP40 [Ratajczak, 1993] and FKBP52 [Ku, 1992] were found to be associated with untransformed steroid receptor heterocomplexes. Both CyP40 and FKBP52 bind to a common site on the steroid receptor-associated heat-shock protein Hsp90. Interaction of FKBP52 with Hsp90 does not occur through its rotamase domain but instead through the tetratricopeptide repeats. FKBP52 is not required for the heterocomplex formation and is instead believed to be important for targeted movement of the receptor. Recent reports have described in more detail the chaperone functions of both CyP-40 [Duina, 1996] and FKBP52 [Bose, 1996] in Hsp90 dependent signal transduction. Immunophilins are involved in several aspects of calcium related cell signaling. FKBP12 regulates intracellular calcium release by interactions with two calcium ion channels, the ryanodine receptor (RyR) and the inositol 1,4,5-trisphosphate receptor (IP3R). RyR, localized to the sarcoplasmic reticulum (SR), is the calcium release channel involved in excitation-contraction coupling in muscle [Fleischer, 1989]. It is also found in other excitable tissue, including the cardiac muscle and brain. It consists of four large (565 kDa) identical subunits, which form the pore. Purification of RyR results in copurification of FKBP12, with one molecule of FKBP12 bound to each of the four 37 Chapter Biological background subunits [Collins, 1991]. Association with FKBP12 stabilizes RyR, improving its Ca2+ fluxing properties [Jayraman, 1992]. FKBP12 may be dissociated from RyR by high concentrations of FK506, implying that FKBP12 associates with RyR through its rotamase domain. Stripped of FKBP12, the calcium fluxing properties of RyR degenerate, and the sarcoplasmic reticulum (SR)/endoplasmic reticulum into the cytosol SR is diminished in its ability to accumulate calcium because pumped in Ca2+ leaks out through RyR. Patch clamp recordings from recombinant RyR indicate that FKBP12 binding has two effects on channel function: it stabilizes both the closed and open states, so that the channel is harder to open, but once open, the Ca2+ fluxing is optimized [Brillantes, 1994]. It is possible that in some cases the immunophilins function as adapter proteins, serving to couple together other macromolecules into assemblies. The rotamase domains may act as "molecular sockets" or recognition domains, with the rotamase activity itself in some cases an artifact. An analogy may be drawn between FKBP and cyclophilin domains and other known adapter molecule domains such as the SH2 and SH3 domains of Src. The recent discovery of molecules that contain both FKBP and cyclophilin domains echoes adapter molecules such as Grb2, which contain both the SH2 and SH3 domains. It is possible that immunophilin-like domains will be found in other proteins not thought of as immunophilins. In this vein, it is interesting to note that the scaffolding protein AKAP78, which anchors both protein kinase A and calcineurin and targets them to sub cellular sites, contains a putative calcineurin binding domain, which resembles FKBP12. 38 Chapter Biological background The high level of conservation and ubiquitous distribution of immunophilins among divergent organisms and in almost all the sub cellular compartments suggest that these proteins participate in important cellular processes. 2.4.2 The immunophilin PPIase activity The broad distribution of the TPR domain immunophilins and the presence of more than one member of the family in most cells suggest that their function(s) is important for cell homeostasis and that there may be redundancy in their action(s). The presence of the PPIase domain leads naturally to the proposal that the action of the Hsp90 binding immunophilins is due to isomerization of prolyl peptide bonds. The PPIase domain of each family is highly conserved between species, from E.coli to humans, although members within each family can diverge greatly outside the PPIase domain. Motifs lying outside the PPIase domain are thought to be responsible for substrate or ligand specificity, but all are thought to be involved in protein folding or protein-protein interactions because of their common PPIase ability. Despite their functional similarity, however, there are few structural similarities between the three classes [Ranganathan et al., 1997]. To date, more than twenty PPIase genes have been characterized in humans. Each has a unique tissue distribution and cellular localization profile. As yet, however, little is known about the functions of these proteins in vivo, although some natural ligands have been identified. A large number of immunophilins belonging to the FKBP and cyclophilin families have been discovered in the past several years, including over 30 cyclophilins 39 Chapter Biological background and more than 20 FKBPs. FKBP immunophilins known to be present in humans include FKBP 12, 12.6, 13, 25, 37, and 52 (by convention, members of the FKBP family are named by appending to the prefix FKBP the apparent molecular weight in kilo Daltons). Cyclophilins found in human tissue include CyP A, B, C, and D, CyP40, and CyP-NK. 2.4.3 Immunophilins and protein folding Their attendant rotamase activity led to the suggestion that immunophilins facilitate protein folding in vivo. Evidence for this hypothesis is accumulating. In one case, CsA, a specific inhibitor for the rotamase activity of cyclophilins, delays the collagen triple helix assembly in chick embryo fibroblasts [Steimann et al., 1991]. The formation of the correct form of transferrin in liver cells is also inhibited by CsA [Lodish and Kong, 1991]. It is not possible, however, to know whether this correlation is due to an important role for the enzymatic activity or whether the enzyme active site is playing the role of a receptor that can bind to unfolded intermediates of protein substrates. 2.5 IMMUNOPHILINS IN HIGHER PLANTS In an attempt to isolate meristem-specific genes, Gasser et al. (1990) isolated a highly abundant transcript from Arabidopsis encoding a cyclophilin. Presence of immunophilins was also supported by the identification of CsA- and FK506-sensitive rotamase activity in plant organelles [Breiman et al., 1992]. Luan et al. (1993) unexpectedly found a CsA- and FK506-sensitive process in plant cells, suggesting the presence of both CYPs and FKBPs in higher plants. Using immunosupressants as an 40 Chapter Biological background affinity tool, multiple CYPs and FKBPs have been purified from various sub cellular compartments in a higher plant [Luan et al., 1993 & 1994a]. The most unique members of plant immunophilins are those that are localized in the chloroplast [Luan et al., 1994a]. Along the way to characterizing plant immunophilins, a number of cyclophilin genes have been characterized [Gasser et al., 1990; Lippuner et al., 1994; Luan et al., 1994b; Marivet et al., 1995; Chou and Gasser, 1997]. Among them, pCyPB/ROC4 encodes a chloroplast cyclophilin [Lippuner et al., 1994; Luan et al., 1994b]. The first plant FKBP-type immunophilin (FKBP15) was cloned from both Arabidopsis and fava bean (Vicia faba) and was shown to be located in the endoplasmic reticulum [Luan et al., 1996]. There are at least two isoforms of FKBP15 in Arabidopsis and they are responsive to heat shock, consistent with the finding on the ER form of FKBP in yeast [Partaledis and Berlin, 1993; Sykes et al., 1993]. High Mr FKBP members have been identified from wheat (Triticum aestivum) and Arabidopsis [Blecher et al., 1996; Vucich and Gasser, 1996; Kurek et al.,1999]. These large FKBPs contain the putative domains for interaction with Hsp90, as reported for FKBP59 and CyP40 in animals [Pratt, 1998]. Recent studies have begun to address the function of immunophilins in plants. The Arabidopsis genome reveals at least 52 genes encoding putative immunophilins and genes encoding parvulin (Harrar et al., 2001). 2.6 ROLE OF PLANT IMMUNOPHILINS As in animals, different members of immunophilins appear to play different roles in plants. In plants, proteins interacting with FKBP12 in the absence or presence of a drug 41 Chapter Biological background are largely unknown. Only the TOR gene of Arabidopsis has been the focus of functional analysis in planta, which shows that the disruption of AtTOR leads to embryo lethality [Menand et al., 2002]. AtFIP37 is critical for embryo and endosperm development and is involved in the endoreplicative cell cycle. Some plant cyclophilin genes have been shown to be induced by a variety of biotic and abiotic stresses, suggesting that they may play a role in environmental response processes [Chou and Gasser, 1997]. An Arabidopsis mutant, pas1, displays abnormal developmental pattern. The PAS1 gene was shown to encode a high Mr FKBP [Vittorioso et al., 1998]. A cyclophilin40 homolog has been shown to regulate the development of leaf shape in Arabidopsis [Berardini et al., 2001]. Several plant cyclophilins interact with an endonuclease involved in T-DNA transfer from agrobacterium to host plant cells [Deng et al., 1998]. Disruption of AtFKBP42 gene function causes developmental defects [Kamphausen et al., 2002]. A chloroplast FKBP has been shown to interact with a photosynthetic electron carrier and affects the accumulation of the protein [Gupta et al., 2002]. The most striking finding is that the Arabidopsis genome encodes 16 chloroplast immunophilin isoforms. While a single cyclophilin is located in the stroma, cyclophilins and FKBPs are targeted to the thylakoid lumen. Experimental corroboration of the genomic data has been carried out for most of these isoforms [Gupta et al., 2002; Peltier et al, 2002; Schubert et al., 2002]. Whilst two of the lumenal immunophilins have been shown to be functionally associated with photosynthetic complex components [Fulgosi et al., 1998; Gupta et al., 2002], the specific function of the majority of chloroplast isoforms remains to be elucidated. 42 Chapter Biological background 2.6.1 Immunophilins and signal transduction in plants Despite the recent progress made on signal transduction pathways in animal and yeast systems, we know very little about the molecular basis of intracellular signaling in plants. Elucidation of signaling pathways inhibited by CsA, FK506, and rapamycin in T cells has established the drugs as powerful tools for studying intracellular signaling in eukaryotic systems. In higher plants, as in animal systems, Ca2+ serves as an important secondary messenger in response to many extracellular stimuli. But the molecular mechanism for Ca2+ function is poorly understood. A good cellular model for studying Ca2+ signaling is stomatal regulation by the plant hormone abscisic acid (ABA). It has been shown that ABA increases the cytoplasmic Ca2+ which in turn inactivates an inward K+ channel (Ikin). Inactivation of Ikin reduces the guard cell turgor and thereby inhibits stomatal opening. Using the patch-clamp technique Luan et al. (1993) have demonstrated that this Ca2+-dependent signaling pathway is blocked by the CsA-CyP and FKBP12FK506 complexes, implicating a calcineurin-like protein phosphatase that mediates the Ca2+ signal in K+ channel regulation in guard cells. Identification of Ca2+-dependent, CsA- and FKBP12-FK506-sensitive protein phosphatase activity from plant protein extract, implicated a Ca2+-dependent protein phosphatase as an important signaling molecule in higher plants. 2.7 IMMUNOPHILINS IN CHLOROPLAST Breiman et al. (1992) found cyclosporin A-sensitive PPIase in the chloroplast. The corresponding gene for this enzyme was cloned in the nuclear genome of Arabidopsis 43 Chapter Biological background thaliana as a single gene targeted to chloroplast [Lippuner, 1994]. Members of the CyP family were identified in various organelles of plants [Luan, 1994; Hayman, 1994; Saito, 1995 and 1998] and the A. thaliana genome project identified at least 10 isoforms of typical cyclophilins [Kaul, 2000]. However, the physiological significance of CyP in the plant cytoplasm and in the chloroplast has been obscure thus far [Luan, 1994; Sheldon, 1996]. The important function of CyP in the process of T-cell activation for the regulation of immunosuppression in mammalian cells suggests that CyP also has an important physiological role in the plant cell, e.g. in signal transduction. Although CyPs of some eukaryotic organisms have several conserved cysteines, there is still no information available on the role of these cysteines. It was found later that CyP is the potential target protein of chloroplast thioredoxin, Trx [Motohashi, K,2001]. 2.7.1 Thylakoid membrane and photosynthesis Thylakoid membranes in the chloroplast catalyze the fundamental process of photosynthetic energy conversion. Outstanding characteristics of this specialized biomembrane are its dual genetic origin [Herrmann, 1996] and the enormous physiological versatility, in particular its ability to manage short- and long-term changes in the light environment [Andersson and Barber, 1994]. Various mechanisms can regulate the distribution of excitation energy between the two photosystems, and others can convert excess excitation energy into thermal energy. Furthermore, a regulated protein turnover can re-establish function once photo-damage has occurred, or acclimatize the photosynthetic machinery to seasonal changes. A multitude of auxiliary enzymes 44 Chapter Biological background [Andersson, 1992] are involved in these physiological processes. These include, for instance, kinases and phosphatases that control the energy distribution between the two photosystems [Allen, 1992; Gal et al., 1997], a substantial number of proteases that catalyze regulated protein degradation [Adam, 1996; Sokolenko et al., 1997] and various protein components that are required during biogenesis of the photosynthetic multisubunit complexes [Herrmann, 1996; Robinson and Knott, 1996]. The identification of such auxiliary components and their genes, and in particular the elucidation of the signal transducing mechanisms connecting light with physiological responses are central and developing topics of current research in photosynthesis. Reversible protein phosphorylation is a primary means of mediating signal transduction in living organisms. In chloroplast thylakoids, nearly a dozen polypeptides, mostly components of the photosystem II complex and its major chlorophyll a/b-binding protein LHCII, can be phosphorylated reversibly in a redox-controlled manner (Allen, 1992; Gal et al., 1997; Vener et al., 1997). Thylakoid protein phosphorylation ensures the energy balance between the two photosystems (Allen, 1992) and regulates protein turnover during the repair of photo-inhibitory damage to photosystem II (Aro et al., 1993; Andersson and Aro, 1997). Although the physiology of thylakoid protein phosphorylation is well characterized in many respects, surprisingly, little is known about the nature of the components involved, their biogenesis, regulation and enzymology. 45 Chapter 2.8 THIOREDOXIN AND IMMUNOPHILINS 2.8.1 Thioredoxin Biological background Thioredoxins (Trx) form a family of redox proteins that are widespread in many species. They are important factors in the regulation of oxidative stress response by interaction with proteins. Thioredoxin (Trx) has two redox active cysteines (WCGPC) in a conserved domain [Buchanan, 1991; Schürmann and Jacquot, 2000; Buchanan et al., 2002]. In higher plants, three different types of Trxs are known based on their localization in the cell: Trx-f and Trx-m in the chloroplast [Jacquot et al., 1997], and Trx-h in the cytosol [Rivera-Madrid et al. 1995]. In addition, several additional Trx groups are reported based on the whole genome data of Arabidopsis thaliana [Laloi et al., 2001; Meyer et al., 2002]. Trx can modulate the enzyme activity of target proteins by reducing disulfide bonds. In some cases, Trx also acts as a hydrogen donor, for example, to reduce peroxiredoxin (Prx). The reduced form Prx reduces hydrogen peroxide, lipid peroxides, or peroxynitrite and functions as an anti-oxidative stress system in the cell [Rouhier and Jacquot 2002].Thus Trx acts as an electron donor to various enzymes. The specificity of Trxs for their targets in the chloroplast is reported to be determined mainly by the surface charges around the active site [Mora-Garcia et al., 1998; Capitani et al., 2000; Collin et al., 2003]. 46 Chapter 2.8.2 Biological background Thioredoxin and redox regulation of immunophilns In the chloroplast of higher plants two Trx isoforms, designated f-type (Trx-f) and m-type (Trx-m) based on their first identified target proteins, are well known [Schürmann, 2000; Jacquot, 1997; Ruelland, 1999]. Various chloroplast enzymes are regulated by reduction of their internal disulfide bonds or reoxidation of thiols. The Calvin cycle enzymes, glyceraldehyde-3-phosphate dehydrogenase, fructose-1,6bisphosphatase, sedoheptulose-1,7-bisphosphatase, and phosphoribulokinase, are regulated by their redox states, and their activities are enhanced in the reduced enzymes [Baumann, 2002]. From this point of view, thioredoxin catalysis controls the process of photosynthesis in plants. Recently several approaches to identify the target proteins of Trx have been challenged [Motohashi, 2001; Yano, 2001; Balmer, 2003]. Within the captured candidate proteins, CyP was identified as an unreported Trx target in the chloroplast [Motohashi, 2001].CyP in the chloroplast is an actual target protein of Trx and that the PPIase activity of CyP is regulated by the reduction of the internal disulfide bond by Trx-m. In addition, the cysteine residues involved in this redox regulation have also been identified [Motohashi, 2003]. Evidence accumulated during the past three decades has shown that chloroplast enzymes are differentially regulated by light in a manner compatible with their function [Buchanan, 1980; Schürmann and Jacquot, 2000]. Thus biosynthetic enzymes, such as the regulatory members of the Calvin cycle, are post-translationally converted from a less active disulfide (S-S) state in the dark to a more active sulfhydryl (SH) state by light 47 Chapter Biological background (Scheme 2. 1). By contrast, other enzymes, e.g., those linked to carbohydrate degradation, behave in the opposite manner, i.e., they are converted from a more active (S-S) state in the dark to a less active (SH) state in the light [Scheme 2.2]. Biosynthetic Enzymeox LIGHT Stroma Biosynthetic Enzymered Stroma Less Active More Active (S-S) (HS-SH) Degradatory Enzymeox LIGHT (Scheme 2.1) Degradatory Enzymered Stroma Stroma More Active Less Active (S-S) (HS-SH) (Scheme 2.2) In this way, chloroplasts use light to maximize the available resources by directing biochemical processes diurnally in an effective manner. The ferredoxin/thioredoxin system—composed of ferredoxin, ferredoxin-thioredoxin reductase and thioredoxin— facilitates these redox changes in the target enzymes that so far are either located in, or exposed to, the stroma. Recent advances in proteomics and genomics have led to the unexpected finding that the lumen, the site of oxygen evolution in chloroplast thylakoids, contains numerous enzymes in addition to those directly associated with the light reactions (Gupta et al., 2002; Peltier et al., 2002; Schubert et al., 2002; Spetea et al., 2004). Prominent among the lumen inhabitants are more than a dozen immunophilins (Gupta et al., 2002; Schubert et 48 Chapter Biological background al., 2002; Peltier et al., 2002; He and Luan, 2004; Romano et al., 2004a), proteins originally defined as receptors for immunosuppressive drugs (Handschumacher et al., 1984) that were later found to have peptidyl-prolyl isomerase (PPIase) activity [Fischer et al., 1989; Schreiber, 1991]. 2.9 THREE-DIMENSIONAL STRUCTURE OF FKBP A number of FKBP X-ray structures have been published including complexes with FK506 [Van Duyne et al., 1993], rapamycin [Van Duyne et al., 1993], a number of piperidinyl derivatives [Holt et al., 1993] and urea-based inhibitors [Choi et al., 1996]. The native FKBP structure [Michnick et al., 1991] and a complex with ascomycin have also been determined by the NMR techniques [Petros et al., 1991]. Structural studies of FKBP12 and cyclophilin A, and their complexes with assorted ligands, have provided a great deal of detailed information on the structure-activity functions in these proteins. hFKBP12 contains a five-stranded antiparallel β-sheet wrapped with a right-handed twist around a short α-helix, together with flexible connecting loops (Fig. 2-3). Twisting the β-sheet results in the formation of hydrophobic concave and convex surfaces. The β-structure is composed of residues 21-31, 35-38, 46-49, 71-78, and 97107 and an α -helix is formed by residues 57-63. The loops comprising residues 39-45 (which connects the two parts of the third β-strand) and 84-91, termed the 40s and 80s loop, respectively, surround the rotamase domain and are rather disordered in the unliganded protein. 49 Chapter Biological background Figure 2-3. The secondary structure elements, including the flap, in the structure of human FKBP-12. The protein is color-coded to mark the residues that are flexible in red, and the regions that are more rigid are shown in white Cyclophilin A, on the other hand, comprises a β-barrel formed by eight β-strands connected by loops and α-helices at the top and bottom (Fig. 2-4). Although cyclophilin's barrel has some similarity to the transport proteins β-lactoglobulin and retinol-binding protein, its topology is unique, and the center of the barrel is blocked with hydrophobic side chains, precluding ligand binding there. 50 Chapter Biological background Binding of FK506 to FKBP12, or CsA to CyPA, does not cause significant changes in the overall three-dimensional structure of either protein but does allow the identification and analysis of the rotamase active sites. Figure 2-4. The structure of Cyclophilin A. The protein is represented in the ribbon form with secondary structure elements α-helices (red), βstrands (yellow arrows), and a 310 helix (cyan ribbon). The loop segments are numbered to 5. 51 Chapter Biological background 2.9.1 Key residues involved in FK506 binding The FK506-binding domain of FKBP12 is a hydrophobic pocket, approximately 9×9 Å in area and Å deep, formed by a convex portion of the β-sheet and three of the loops. In hFKBP12, the substrate-binding pocket for the pipecolinylring of FK506 has been identified, which consists of Tyr26, Phe36, Asp37, Phe46, Phe48, Val55, Ile56, Trp59, Tyr82, Ile90, Ile91, Leu97, and Phe99.The FKBP12 binding portion of FK506 comprises the pipecolinyl ring together with the pendant pyranose ring and ketoamide linkage and a portion of the cyclohexylpropenyl ester side chain. Two hydrophobic pockets in the FKBP12 binding domain are occupied by FK506: (1) a pocket defined by Trp59 (the "floor") and the side chains of Tyr26, Phe46, Phe99, Val55, and Ile56 (the "walls"), into which the pipecolinyl ring binds deeply, and (2) another hydrophobic cavity formed by the side chains of Ile90, Ile91, His87, Phe36, Tyr82, and Asp37, into which the pyranose ring binds. In addition to the complex with FK506, the structures of FKBP12 complex with the related immunosuppressant drug ascomycin [Meadows, 1993], rapamycin [Van Duyne, 1993; Wilson, 1995], a nonimmunosuppressive analogue of ascomycin, 18hydroxyascomycin (or L-685,818, Becker, 1993}, and several small molecular inhibitors [Holt, 1993] have been determined. The FKBP binding domain portions of all these molecules are closely superimposable in all of these structures. The remaining portion of the macrocyclic compounds extends into the solvent-accessible region of the protein and is crucial for mediating the immunosuppressive effects of the drug-immunophilin complexes. 52 Chapter 2.9.2 Biological background Catalytic domain The residues which make up the FK506 binding site of hFKBP12 define a common "FKBP domain", which is remarkably conserved both across species and in higher molecular weight members of the FKBP family [Kay, 1996]. Ten residues are strongly conserved, and Tyr26, Phe36, Asp37, Val55, Ile56, Tyr82, and Phe99 are completely conserved in all known FKBPs (all species and isoforms) with significant PPIase activity. The greatest variation in the domain is found in His87, which is replaced by a variety of hydrophobic or hydrophilic residues in other FKBPs. Phe46 may be replaced by other hydrophobic residues with little effect on the rotamase activity. FKBP12.6 differs from FKBP12 in its rotamase domain only by the replacement of Trp59 with Phe, which does not alter appreciably either the rotamase activity or FK506 binding. Mutagenesis experiments on hFKBP12 have demonstrated that the rotamase activity and drug binding may be structurally dissected. The mutation F36Y was much more deleterious to the rotamase activity than to macrolide binding, as was the mutation Y82L; the mutation W59A abolishes the rotamase activity but not FK506 binding. Replacement of Asp37 with Val [Aldape, 1996], or Phe99 with Tyr [Timerman, 1995] completely abolished both the PPIase activity and FK506 binding. 2.9.3 PURPOSE OF ATFKBP13 STRUCTURE DETERMINATION Our recent study on AtFKBP13, a representative of the FKBP immunophilin group and a resident of the chloroplast thylakoid lumen, demonstrates an interaction of 53 Chapter Biological background the enzyme with lumenal redox proteins participating in the photosynthetic electron transfer [Gupta et al., 2002]. Coupled with evidence that a stromal cyclophilin ROC4 (now referred to as AtCYP20-3, He and Luan 2004; Romano et al., 2004b) is activated by thioredoxin [Motohashi et al., 2003], this finding raises the question of whether the regulation of AtFKBP13 could be linked to redox. We have addressed the question by determining the structure of AtFKBP13 in oxidized and reduced states, studying its response to redox agents and later comparing the two structures for disulfide mediated changes. 54 [...]... characterized in many respects, surprisingly, little is known about the nature of the components involved, their biogenesis, regulation and enzymology 45 Chapter 2 2.8 THIOREDOXIN AND IMMUNOPHILINS 2. 8.1 Biological background Thioredoxin Thioredoxins (Trx) form a family of redox proteins that are widespread in many species They are important factors in the regulation of oxidative stress response by interaction... numbered 1 to 5 51 Chapter 2 Biological background 2. 9.1 Key residues involved in FK506 binding The FK506-binding domain of FKBP 12 is a hydrophobic pocket, approximately 9×9 Å in area and 7 Å deep, formed by a convex portion of the β-sheet and three of the loops In hFKBP 12, the substrate-binding pocket for the pipecolinylring of FK506 has been identified, which consists of Tyr26, Phe36, Asp37, Phe46,... cyclophilin ROC4 (now referred to as AtCYP20-3, He and Luan 20 04; Romano et al., 20 04b) is activated by thioredoxin [Motohashi et al., 20 03], this finding raises the question of whether the regulation of AtFKBP13 could be linked to redox We have addressed the question by determining the structure of AtFKBP13 in oxidized and reduced states, studying its response to redox agents and later comparing the... by directing biochemical processes diurnally in an effective manner The ferredoxin/thioredoxin system—composed of ferredoxin, ferredoxin-thioredoxin reductase and thioredoxin— facilitates these redox changes in the target enzymes that so far are either located in, or exposed to, the stroma Recent advances in proteomics and genomics have led to the unexpected finding that the lumen, the site of oxygen... evolution in chloroplast thylakoids, contains numerous enzymes in addition to those directly associated with the light reactions (Gupta et al., 20 02; Peltier et al., 20 02; Schubert et al., 20 02; Spetea et al., 20 04) Prominent among the lumen inhabitants are more than a dozen immunophilins (Gupta et al., 20 02; Schubert et 48 Chapter 2 Biological background al., 20 02; Peltier et al., 20 02; He and Luan, 20 04;... protein [Gupta et al., 20 02] The most striking finding is that the Arabidopsis genome encodes 16 chloroplast immunophilin isoforms While a single cyclophilin is located in the stroma, 5 cyclophilins and 9 FKBPs are targeted to the thylakoid lumen Experimental corroboration of the genomic data has been carried out for most of these isoforms [Gupta et al., 20 02; Peltier et al, 20 02; Schubert et al., 20 02] ... CsA-CyP and FKBP12FK506 complexes, implicating a calcineurin-like protein phosphatase that mediates the Ca2+ signal in K+ channel regulation in guard cells Identification of Ca2+-dependent, CsA- and FKBP 12- FK506-sensitive protein phosphatase activity from plant protein extract, implicated a Ca2+-dependent protein phosphatase as an important signaling molecule in higher plants 2. 7 IMMUNOPHILINS IN CHLOROPLAST... Ile56, Trp59, Tyr 82, Ile90, Ile91, Leu97, and Phe99.The FKBP 12 binding portion of FK506 comprises the pipecolinyl ring together with the pendant pyranose ring and ketoamide linkage and a portion of the cyclohexylpropenyl ester side chain Two hydrophobic pockets in the FKBP 12 binding domain are occupied by FK506: (1) a pocket defined by Trp59 (the "floor") and the side chains of Tyr26, Phe46, Phe99,... functions of these proteins in vivo, although some natural ligands have been identified A large number of immunophilins belonging to the FKBP and cyclophilin families have been discovered in the past several years, including over 30 cyclophilins 39 Chapter 2 Biological background and more than 20 FKBPs FKBP immunophilins known to be present in humans include FKBP 12, 12. 6, 13, 25 , 37, and 52 (by convention,... and Jacquot 20 02] .Thus Trx acts as an electron donor to various enzymes The specificity of Trxs for their targets in the chloroplast is reported to be determined mainly by the surface charges around the active site [Mora-Garcia et al., 1998; Capitani et al., 20 00; Collin et al., 20 03] 46 Chapter 2 2.8 .2 Biological background Thioredoxin and redox regulation of immunophilns In the chloroplast of higher . directing biochemical processes diurnally in an effective manner. The ferredoxin/thioredoxin system—composed of ferredoxin, ferredoxin-thioredoxin reductase and thioredoxin— facilitates these redox. 46 2. 8 THIOREDOXIN AND IMMUNOPHILINS 2. 8.1 Thioredoxin Thioredoxins (Trx) form a family of redox proteins that are widespread in many species. They are important factors in the regulation of oxidative. SH2 and SH3 domains. It is possible that immunophilin-like domains will be found in other proteins not thought of as immunophilins. In this vein, it is interesting to note that the scaffolding