Cyclophilin and peptidyl-prolyl cis-trans isomerase are probably identical proteins

The enzyme peptidyl-prolyl cis-trans isomerase (PPIase) was recently discovered in mammalian tissues and purified from porcine kidney. It catalyses the slow cis-trans isomerization of proline peptide (Xaa-Pro) bonds in oligopeptides and accelerates slow, rate-limiting steps in the folding of several proteins. Here, we report the N-terminal sequence of PPIase together with further chemical and enzymatic properties. The results indicate that this enzyme is probably identical to cyclophilin, a recently discovered mammalian protein which binds tightly to cyclosporin A (CsA). Cyclophilin is thought to be linked to the immunosuppressive action of CsA. The first 38 amino-acid residues of porcine PPIase and of bovine cyclophilin are identical and the two proteins both have a relative molecular mass of about 17,000 (ref. 7). The catalysis of prolyl isomerization in oligopeptides and of protein folding by PPIase are strongly inhibited in the presence of low levels of CsA. The activities of both PPIase and cyclophilin depend on a single sulphydryl group. At present it is unknown whether the inhibition of prolyl isomerase activity is related with the immunosuppressive action of CsA.     

Catalysis of protein folding by prolyl isomerase

Rates of protein folding reactions vary considerably. Some denatured proteins regain the native conformation within milliseconds or seconds, whereas others refold very slowly in the time range of minutes or hours. Varying folding rates are observed not only for different proteins, but can also be detected for single polypeptide species. This originates from the co-existence of fast- and slow-folding forms of the unfolded protein, which regain the native state with different rates. The proline hypothesis provides a plausible explanation for this heterogeneity. It assumes that the slow-folding molecules possess non-native isomers of peptide bonds between proline and another residue, and that crucial steps in the refolding of the slow-folding molecules are limited in rate by the slow reisomerization of such incorrect proline peptide bonds. Recently the enzyme peptidyl-prolyl cis-trans isomerase (PPIase) was discovered and purified from pig kidney. It catalyses efficiently the cis in equilibrium trans isomerization of proline imidic peptide bonds in oligopeptides. Here we show that it also catalyses slow steps in the refolding of a number of proteins of which fast- and slow-folding species have been observed and where it was suggested that proline isomerization was involved in slow refolding. The efficiency of catalysis depends on the accessibility for the isomerase of the particular proline peptide bonds in the refolding protein chain.     

Peptidyl-prolyl cis-trans isomerase is the cyclosporin A-binding protein cyclophilin

Peptidyl-prolyl cis-trans isomerase (PPIase) catalyses the cis-trans isomerization of proline imidic peptide bonds in oligopeptides and has been shown to accelerate the refolding of several proteins in vitro. Its activity has been detected in yeast, insects and Escherichia coli as well as in mammals, and it is though to be essential for protein folding during protein synthesis in the cell. We purified PPIase from pig kidney and found that its amino-acid sequence is identical to that reported for bovine cyclophilin, a protein known to bind the immunosuppressive drug, cyclosporin A (ref. 5). To investigate the functional relationship between PPIase and cyclophilin we examined the effect of cyclosporin A on PPIase activity and found that it was inhibitory. Thus we propose that the peptidyl-prolyl cis-trans isomerizing activity of PPIase may be involved in events, such as those occurring early in T-cell activation, that are suppressed by cyclosporin A.     

Glycoproteins form mixed disulphides with oxidoreductases during folding in living cells

The formation of intra- and interchain disulphide bonds constitutes an integral part of the maturation of most secretory and membrane-bound proteins in the endoplasmic reticulum. Evidence indicates that members of the protein disulphide isomerase (PDI) superfamily are part of the machinery needed for proper oxidation and isomerization of disulphide bonds. Models based on in vitro studies predict that the formation of mixed disulphide bonds between oxidoreductase and substrate is intermediate in the generation of the native intrachain disulphide bond in the substrate polypeptide. Whether this is how thiol oxidoreductases work inside the endoplasmic reticulum is not clear. Nor has it been established which of the many members of the PDI superfamily interacts directly with newly synthesized substrate proteins, because transient mixed disulphides have never been observed in the mammalian endoplasmic reticulum during oxidative protein folding. Here we describe the mechanisms involved in co- and post-translational protein oxidation in vivo. We show that the endoplasmic-reticulum-resident oxidoreductases PDI and ERp57 are directly involved in disulphide oxidation and isomerization, and, together with the lectins calnexin and calreticulin, are central in glycoprotein folding in the endoplasmic reticulum of mammalian cells.     

A receptor for the immunosuppressant FK506 is a cis-trans peptidyl-prolyl isomerase

The structurally novel macrolide FK506 (refs 1,2) has recently been demonstrated to have potent immunosuppressive activity at concentrations several hundredfold lower than cyclosporin A (CsA). Cyclosporin A, a cyclic peptide, has found widespread clinical use in the prevention of graft rejection following bone marrow and organ transplantation. The mechanisms of immunosuppression mediated by FK506 and CsA appear to be remarkably similar, suggesting that these unrelated structures act on a common receptor or on similar molecular targets, perhaps the CsA receptor, cyclophilin, which has recently been shown by Fischer et al. and Takahashi et al. to have cis-trans peptidyl-prolyl isomerase activity. We have prepared an FK506 affinity matrix and purified a binding protein for FK506 from bovine thymus and from human spleen. This FK506-binding protein (FKBP) has a relative molecular mass (Mr) of approximately 14,000(14K), a pI of 8.8-8.9, and does not cross-react with antisera against cyclophilin. The first 40 N-terminal residues of the bovine and 16 residues of the human FKBP were determined; the 16-residue fragments are identical to each other and unrelated to any known sequences. This protein catalyses the cis-trans isomerization of the proline amide in a tetrapeptide substrate and FK506 inhibits the action of this new isomerase. The FKBP and cyclophilin appear to be members of an emerging class of novel proteins that regulate T cell activation and other metabolic processes, perhaps by the recognition (and possibly the isomerization) of proline-containing epitopes in target proteins.     

Sequence of protein disulphide isomerase and implications of its relationship to thioredoxin

The formation of disulphide bonds is essential to the structure and function of proteins. These bonds rapidly form either cotranslationally or immediately post-translationally in the lumen of the endoplasmic reticulum. Native disulphide pairing for such proteins has been achieved in vitro; however, the rates of reassembly are slow and the conditions non-physiological. To account for these observations, Anfinsen et al. proposed that a 'disulphide interchange protein' was the in vivo catalyst of disulphide bond rearrangement. Other groups discovered an activity with similar characteristics that catalysed the reductive cleavage of insulin and may be associated with insulin degradation, although this result has been disputed. The enzyme involved, protein disulphide isomerase (PDI; EC 5.3.4.1), may be the in vivo catalyst of disulphide bond formation. Here we describe the sequence of cloned rat liver PDI complementary DNA which predicts a protein with two distinct regions homologous with Escherichia coli thioredoxin, a known cofactor in oxidation-reduction reactions. Each of these regions contains the presumed active site sequence Trp-Cys-Gly-His-Cys-Lys, suggesting that PDI, similar in action to thioredoxin, catalyses disulphide bond interchange via an internal disulphide-sulphydryl interchange. The cDNA predicts a signal peptide consistent with the view that PDI is a luminal endoplasmic reticulum protein. PDI messenger RNA, although ubiquitous, is more highly concentrated in secretory cells.     

Defective co-translational formation of disulphide bonds in protein disulphide-isomerase-deficient microsomes

The formation of disulphide bonds in mammalian secretory and cell-surface proteins occurs in the lumen of the endoplasmic reticulum and is believed to be catalysed by the enzyme protein disulphide-isomerase (PDI). The evidence for this physiological role for PDI is circumstantial and relates to the cell and tissue distribution of the enzyme, its developmental behaviour and its catalytic properties in vitro. A clear requirement for PDI in the correct folding or assembly of disulphide-bonded proteins during biosynthesis has not been demonstrated. We have prepared dog pancreas microsomes which are deficient in soluble lumenal proteins, including PDI, but which are still able to translocate and process proteins synthesized in vitro. Using the formation of intramolecular disulphide bonds during the in vitro synthesis of gamma-gliadin, a wheat storage protein, as a model, we have demonstrated that these microsomes are defective in co-translational formation of disulphide bonds. Reconstitution of these microsomes with purified PDI reverses this defect.     

The prolyl isomerase Pin1 restores the function of Alzheimer-associated phosphorylated tau protein.

One of the neuropathological hallmarks of Alzheimer's disease is the neurofibrillary tangle, which contains paired helical filaments (PHFs) composed of the microtubule-associated protein tau. Tau is hyperphosphorylated in PHFs, and phosphorylation of tau abolishes its ability to bind microtubules and promote microtubule assembly. Restoring the function of phosphorylated tau might prevent or reverse PHF formation in Alzheimer's disease. Phosphorylation on a serine or threonine that precedes proline (pS/T-P) alters the rate of prolyl isomerization and creates a binding site for the WW domain of the prolyl isomerase Pin1. Pin1 specifically isomerizes pS/T-P bonds and regulates the function of mitotic phosphoproteins. Here we show that Pin1 binds to only one pT-P motif in tau and copurifies with PHFs, resulting in depletion of soluble Pin1 in the brains of Alzheimer's disease patients. Pin1 can restore the ability of phosphorylated tau to bind microtubules and promote microtubule assembly in vitro. As depletion of Pin1 induces mitotic arrest and apoptotic cell death, sequestration of Pin1 into PHFs may contribute to neuronal death. These findings provide a new insight into the pathogenesis of Alzheimer's disease.     

Intrinsic dynamics of an enzyme underlies catalysis

A unique feature of chemical catalysis mediated by enzymes is that the catalytically reactive atoms are embedded within a folded protein. Although current understanding of enzyme function has been focused on the chemical reactions and static three-dimensional structures, the dynamic nature of proteins has been proposed to have a function in catalysis. The concept of conformational substates has been described; however, the challenge is to unravel the intimate linkage between protein flexibility and enzymatic function. Here we show that the intrinsic plasticity of the protein is a key characteristic of catalysis. The dynamics of the prolyl cis-trans isomerase cyclophilin A (CypA) in its substrate-free state and during catalysis were characterized with NMR relaxation experiments. The characteristic enzyme motions detected during catalysis are already present in the free enzyme with frequencies corresponding to the catalytic turnover rates. This correlation suggests that the protein motions necessary for catalysis are an intrinsic property of the enzyme and may even limit the overall turnover rate. Motion is localized not only to the active site but also to a wider dynamic network. Whereas coupled networks in proteins have been proposed previously, we experimentally measured the collective nature of motions with the use of mutant forms of CypA. We propose that the pre-existence of collective dynamics in enzymes before catalysis is a common feature of biocatalysts and that proteins have evolved under synergistic pressure between structure and dynamics.     

The prolyl isomerase Pin1 regulates amyloid precursor protein processing and amyloid-beta production

Neuropathological hallmarks of Alzheimer's disease are neurofibrillary tangles composed of tau and neuritic plaques comprising amyloid-beta peptides (Abeta) derived from amyloid precursor protein (APP), but their exact relationship remains elusive. Phosphorylation of tau and APP on certain serine or threonine residues preceding proline affects tangle formation and Abeta production in vitro. Phosphorylated Ser/Thr-Pro motifs in peptides can exist in cis or trans conformations, the conversion of which is catalysed by the Pin1 prolyl isomerase. Pin1 has been proposed to regulate protein function by accelerating conformational changes, but such activity has never been visualized and the biological and pathological significance of Pin1 substrate conformations is unknown. Notably, Pin1 is downregulated and/or inhibited by oxidation in Alzheimer's disease neurons, Pin1 knockout causes tauopathy and neurodegeneration, and Pin1 promoter polymorphisms appear to associate with reduced Pin1 levels and increased risk for late-onset Alzheimer's disease. However, the role of Pin1 in APP processing and Abeta production is unknown. Here we show that Pin1 has profound effects on APP processing and Abeta production. We find that Pin1 binds to the phosphorylated Thr 668-Pro motif in APP and accelerates its isomerization by over 1,000-fold, regulating the APP intracellular domain between two conformations, as visualized by NMR. Whereas Pin1 overexpression reduces Abeta secretion from cell cultures, knockout of Pin1 increases its secretion. Pin1 knockout alone or in combination with overexpression of mutant APP in mice increases amyloidogenic APP processing and selectively elevates insoluble Abeta42 (a major toxic species) in brains in an age-dependent manner, with Abeta42 being prominently localized to multivesicular bodies of neurons, as shown in Alzheimer's disease before plaque pathology. Thus, Pin1-catalysed prolyl isomerization is a novel mechanism to regulate APP processing and Abeta production, and its deregulation may link both tangle and plaque pathologies. These findings provide new insight into the pathogenesis and treatment of Alzheimer's disease.     

The ninaA gene required for visual transduction in Drosophila encodes a homologue of cyclosporin A-binding protein

Mutations of the Drosophila melanogaster ninaA gene affect phototransduction: ninaA mutant flies have a 10-fold reduction in the levels of rhodopsin in the R1-R6 photoreceptor cells. The ninaA gene was isolated and found to encode a 237-amino-acid protein that has over 40% amino-acid sequence identity with the vertebrate cyclosporin A-binding protein, cyclophilin, a protein that seems to be involved in T-lymphocyte activation. The remarkable evolutionary conservation of cyclophilin in two phylogenetically distant organisms and its involvement in diverse transduction processes suggests that this protein plays an important role in cellular metabolism. Indeed, cyclophilin has recently been shown to be a prolyl cis-trans isomerase that catalyses, in vitro, rate-limiting steps in the folding of a number of proteins. Here, we present evidence for the involvement of cyclophilin-like molecules in a defined cellular process. The availability of mutations in a cyclophilin gene provides a new model system for the study of cyclophilin and cyclosporin action.     

Cis-trans isomerization at a proline opens the pore of a neurotransmitter-gated ion channel

5-hydroxytryptamine type 3 (5-HT3) receptors are members of the Cys-loop receptor superfamily. Neurotransmitter binding in these proteins triggers the opening (gating) of an ion channel by means of an as-yet-uncharacterized conformational change. Here we show that a specific proline (Pro 8*), located at the apex of the loop between the second and third transmembrane helices (M2-M3), can link binding to gating through a cis-trans isomerization of the protein backbone. Using unnatural amino acid mutagenesis, a series of proline analogues with varying preference for the cis conformer was incorporated at the 8* position. Proline analogues that strongly favour the trans conformer produced non-functional channels. Among the functional mutants there was a strong correlation between the intrinsic cis-trans energy gap of the proline analogue and the activation of the channel, suggesting that cis-trans isomerization of this single proline provides the switch that interconverts the open and closed states of the channel. Consistent with this proposal, nuclear magnetic resonance studies on an M2-M3 loop peptide reveal two distinct, structured forms. Our results thus confirm the structure of the M2-M3 loop and the critical role of Pro 8* in the 5-HT3 receptor. In addition, they suggest that a molecular rearrangement at Pro 8* is the structural mechanism that opens the receptor pore.     

补阳还五汤与高血压病气虚血瘀证“方证相关”的蛋白质组学研究

揭示补阳还五汤与高血压病气虚血瘀证"方证相关"的现代生物学基础,制作高血压病气虚血瘀证细胞模型。用补阳还五汤含药血清干预细胞模型,利用双向凝胶电泳(2-DE)及基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS/MS)技术筛选及鉴定差异表达的蛋白点。并对其进行生物学分析。结果高血压病气虚血瘀证组与健康对照组比较,差异蛋白质点有30个。其中,有16个蛋白上调,14个蛋白质下调。补阳还五汤组与高血压病气虚血瘀证组比较,差异蛋白质点有14个。其中,有9个蛋白上调,5个蛋白质下调。MALDI-TOF-MS/MS鉴定出:高血压病气虚血瘀证组与健康对照组差异蛋白点共有8个蛋白被成功鉴定出来。表达上调的蛋白有肽基脯氨酰异构酶A、肽基脯氨酸顺反异构酶A样1亚型、真核翻译起始因子5A-1 B亚型、微管蛋白beta-2C、CRA_b亚型、3-羟酰辅酶A脱氢酶2型异构体2。表达下调的蛋白有丙酮酸激酶同工酶M1亚型、抑制蛋白-1、抑制蛋白-1 1亚型、补阳还五汤组与高血压病气虚血瘀证组差异蛋白点共有3个蛋白被成功鉴定出来。表达上调的蛋白有丙酮酸激酶CRA_c亚型、热休克蛋白27;表达下调的蛋白有膜联蛋白A1,CRA_b亚型。这些蛋白多与促进或抑制细胞凋亡有关。说明高血压病气虚血瘀证存在着细胞凋亡,补阳还五汤可以纠正高血压病气虚血瘀证引起的细胞凋亡。这些差异蛋白可以作为高血压病气虚血瘀证的标志蛋白或补阳还五汤的作用靶点,抑制细胞凋亡可能是补阳还五汤与高血压病气虚血瘀证"方证相关"的现代生物学基础之一。     

Role of the prolyl isomerase Pin1 in protecting against age-dependent neurodegeneration

The neuropathological hallmarks of Alzheimer's disease and other tauopathies include senile plaques and/or neurofibrillary tangles. Although mouse models have been created by overexpressing specific proteins including beta-amyloid precursor protein, presenilin and tau, no model has been generated by gene knockout. Phosphorylation of tau and other proteins on serine or threonine residues preceding proline seems to precede tangle formation and neurodegeneration in Alzheimer's disease. Notably, these phospho(Ser/Thr)-Pro motifs exist in two distinct conformations, whose conversion in some proteins is catalysed by the Pin1 prolyl isomerase. Pin1 activity can directly restore the conformation and function of phosphorylated tau or it can do so indirectly by promoting its dephosphorylation, which suggests that Pin1 is involved in neurodegeneration; however, genetic evidence is lacking. Here we show that Pin1 expression is inversely correlated with predicted neuronal vulnerability and actual neurofibrillary degeneration in Alzheimer's disease. Pin1 knockout in mice causes progressive age-dependent neuropathy characterized by motor and behavioural deficits, tau hyperphosphorylation, tau filament formation and neuronal degeneration. Thus, Pin1 is pivotal in protecting against age-dependent neurodegeneration, providing insight into the pathogenesis and treatment of Alzheimer's disease and other tauopathies.     

Sensitivity to cyclosporin A is mediated by cyclophilin in Neurospora crassa and Saccharomyces cerevisiae

Cyclosporin A, a cyclic fungal undecapeptide produced by Tolypocladium inflatum, is a potent immunosuppressive drug originally isolated as an antifungal antibiotic. Cyclosporin A (CsA) is widely used in humans to prevent rejection of transplanted organs such as kidney, heart, bone marrow and liver. The biochemical basis of CsA action is not known: its primary cellular target has been suggested to be calmodulin, the prolactin receptor or cyclophilin, a CsA-binding protein originally isolated from the cytosol of bovine thymocytes. Cyclophilin has been shown to be a highly conserved protein present in all eukaryotic cells tested and to be identical to peptidyl-prolyl cis-trans isomerase, a novel type of enzyme that accelerates the slow refolding phase of certain proteins in vitro. We demonstrate that in the lower eukaryotes N. crassa and S. cerevisiae, cyclo philin mediates the cytotoxic CsA effect. In CsA-resistant mutants of both organisms, the cyclophilin protein is either lost completely or, if present, has lost its ability to bind CsA.     

The molten globule protein conformation probed by disulphide bonds

The molten globule is a compact protein conformation that has a secondary structure content like that of the native protein, but poorly defined tertiary structure. It is a stable state for a few proteins under particular conditions and could be a ubiquitous kinetic intermediate in protein folding. The extent to which native interactions, above the level of the secondary structure, are preserved in this conformation is not so far known. Here we report that alpha-lactalbumin can adopt a molten globule conformation when one of its four disulphide bonds is reduced. In this state, the three other disulphide bonds rearrange spontaneously, at the same rate as when the protein is fully unfolded, to a number of different disulphide bond isomers that tend to maintain the molten globule conformation. That the molten globule state is compatible with a variety of disulphide bond pairings suggests that it is unlikely to be stabilized by many specific tertiary interactions.     

Role of ATP and disulphide bonds during protein folding in the endoplasmic reticulum.

Being topologically equivalent to the extracellular space, the lumen of the endoplasmic reticulum (ER) provides a unique folding environment for newly synthesized proteins. Unlike other compartments in the cell where folding occurs, the ER is oxidizing and therefore can promote the formation of disulphide bonds. The reducing agent dithiothreitol, when added to living cells, inhibits disulphide formation with profound effects on folding. Taking advantage of this effect, we demonstrate here that folding of influenza haemagglutinin is energy dependent. Metabolic energy is required to support the correct folding and disulphide bond formation in this well characterized viral glycoprotein, to rescue misfolded proteins from disulphide-linked aggregates, and to maintain the oxidized protein in its folded and oligomerization-competent state.     

The dynamic disulphide relay of quiescin sulphydryl oxidase

Protein stability, assembly, localization and regulation often depend on the formation of disulphide crosslinks between cysteine side chains. Enzymes known as sulphydryl oxidases catalyse de novo disulphide formation and initiate intra- and intermolecular dithiol/disulphide relays to deliver the disulphides to substrate proteins. Quiescin sulphydryl oxidase (QSOX) is a unique, multi-domain disulphide catalyst that is localized primarily to the Golgi apparatus and secreted fluids and has attracted attention owing to its overproduction in tumours. In addition to its physiological importance, QSOX is a mechanistically intriguing enzyme, encompassing functions typically carried out by a series of proteins in other disulphide-formation pathways. How disulphides are relayed through the multiple redox-active sites of QSOX and whether there is a functional benefit to concatenating these sites on a single polypeptide are open questions. Here we present the first crystal structure of an intact QSOX enzyme, derived from a trypanosome parasite. Notably, sequential sites in the disulphide relay were found more than 40?? apart in this structure, too far for direct disulphide transfer. To resolve this puzzle, we trapped and crystallized an intermediate in the disulphide hand-off, which showed a 165° domain rotation relative to the original structure, bringing the two active sites within disulphide-bonding distance. The comparable structure of a mammalian QSOX enzyme, also presented here, shows further biochemical features that facilitate disulphide transfer in metazoan orthologues. Finally, we quantified the contribution of concatenation to QSOX activity, providing general lessons for the understanding of multi-domain enzymes and the design of new catalytic relays.     

Trigger factor and DnaK cooperate in folding of newly synthesized proteins.

The role of molecular chaperones in assisting the folding of newly synthesized proteins in the cytosol is poorly understood. In Escherichia coli, GroEL assists folding of only a minority of proteins and the Hsp70 homologue DnaK is not essential for protein folding or cell viability at intermediate growth temperatures. The major protein associated with nascent polypeptides is ribosome-bound trigger factor, which displays chaperone and prolyl isomerase activities in vitro. Here we show that delta tig::kan mutants lacking trigger factor have no defects in growth or protein folding. However, combined delta tig::kan and delta dnaK mutations cause synthetic lethality. Depletion of DnaK in the delta tig::kan mutant results in massive aggregation of cytosolic proteins. In delta tig::kan cells, an increased amount of newly synthesized proteins associated transiently with DnaK. These findings show in vivo activity for a ribosome-associated chaperone, trigger factor, in general protein folding, and functional cooperation of this protein with a cytosolic Hsp70. Trigger factor and DnaK cooperate to promote proper folding of a variety of E. coli proteins, but neither is essential for folding and viability at intermediate growth temperatures.     

Role of a subdomain in the folding of bovine pancreatic trypsin inhibitor.

The disulphide-bonded intermediates that accumulate in the oxidative folding of bovine pancreatic trypsin inhibitor (BPTI) were characterized some time ago. Structural characterization of these intermediates would provide an explanation of the kinetically preferred pathways of folding for BPTI. When folding occurs under strongly oxidizing conditions, more than half the molecules become trapped in an intermediate, designated N*, which is similar to the native protein but lacks the 30-51 disulphide bond. We have tested the hypothesis that the precursor to N* is the one-disulphide intermediate [5-55], which contains the most stable disulphide in BPTI, and present evidence here that this is the case. A peptide model of [5-55], corresponding to a subdomain of BPTI, seems to fold into a native-like conformation, explaining why [5-55] does not lead to native protein and why it folds rapidly to N*. A native-like subdomain structure in a peptide model of [30-51], the other crucial one-disulphide intermediate, may explain the route by which [30-51] folds to native protein. Thus, much of the folding pathway of BPTI can be explained by the formation of a native-like subdomain in these two early intermediates. This suggests that a large part of the protein folding problem can be reduced to identifying and understanding subdomains of native proteins.