Structural investigations of redox regulation in ATFKBP13 2

Fischer termed this enzymatic activity peptidylprolyl trans isomerase activity, and the enzyme became known as PPIase [Fischer, 1984]... Immunosuppressant drugs which are selective inhib

Chapter 2 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 trans isomerase activity, and the enzyme became known as PPIase [Fischer, 1984]

cis-Chapter 2 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)

Chapter 2 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

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

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,

Chapter 2 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 IL-2-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 glutamate-induced 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

Chapter 2 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 do 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

Chapter 2 Biological background

ryanodine and inositol 1,4,5-triphosphate receptors), which is disrupted by FK506, thereby increasing Ca2+ flux; the type 1 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]

To understand the molecular mechanisms of immunosuppression by CsA, FK506, and rapamycin, the cellular receptors of these drugs have been purified and characterized

Chapter 2 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]

of calcineurin, a Ca2+/calmodulin-dependent protein phosphatase, thereby blocking the

Chapter 2 Biological background

signaling pathway for T-cell activation [Liu et al., 1991; Clipstoneand Crabtree, 1992]

these immunophilins affect receptor action or heterocomplex assembly in any way

Owens-Grillo et al., 1996a]

protein-proteininteractions [Sikorski et al., 1990] The immunophilins in steroid-receptor

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

Chapter 2 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

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

Chapter 2 Biological background

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

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,

[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

Chapter 2 Biological background

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

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, 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

In an attempt to isolate meristem-specific genes, Gasser etal (1990) isolated a

rotamase activityin plant organelles [Breiman et al., 1992] Luan et al (1993)

Chapter 2 Biological background

compartmentsin a higher plant [Luan et al., 1993 & 1994a] Themost unique members of plant immunophilins are those that arelocalized in the chloroplast [Luan et al., 1994a] Along theway to characterizing plant immunophilins, a number of cyclophilingenes have been characterized [Gasser et al., 1990; Lippuneret al., 1994; Luan et al., 1994b; Marivet

and was shown tobe located in the endoplasmic reticulum [Luan et al., 1996] There are

consistent with the finding on the ER form of FKBP in yeast [Partaledis and Berlin, 1993;

aestivum) and Arabidopsis [Blecher et al., 1996; Vucich and Gasser, 1996; Kurek et

al.,1999] These large FKBPs contain the putative domains for interactionwith Hsp90, as

address the function ofimmunophilins in plants The Arabidopsis genome reveals at least

2001)

As in animals, differentmembers of immunophilins appear to play different roles

in plants.In plants, proteins interacting with FKBP12 in the absence orpresence of a drug