Trans Isomerase Research Articles

Pin1 affects Tau phosphorylation in response to Aβ oligomers

We show that in hippocampal cultured neurons, dephosphorylation of peptidyl–prolyl cis–trans isomerase Pin1 on Ser16 is occurring during the early stages of exposure to Aβ (1–42) oligomers. This occurrence, resulting in Pin1 activation, is paralleled by Tau Thr231 dephosphorylation, probably due to Pin1-mediated Tau isomerisation. Indeed, in the presence of the specific Pin1 inhibitor juglone, Aβ-induced Tau Thr231dephosphorylation is prevented. The involvement of protein phosphatase 2A (PP2A) in dephosphorylation of isomerised Tau is shown by the co-treatment of neurons with Aβ (1–42) and okadaic acid, a PP2A inhibitor, leading to Tau Thr231 hyperphosphorylation. We also report the modulation, via Pin1, of Ser199, Ser396, Ser400 and Ser404 phosphorylation state in response to Aβ treatment. Taken together, these data suggest for the first time that an early Pin1 response might be transiently evoked by Aβ 1–42 oligomers, preventing Tau hyperphosphorylation. This evidence highlights the role of Pin1 as Tau phosphorylation modulator during Alzheimer onset.

Differential composition of culture supernatants from wild-type Brucella abortus and its isogenic virB mutants

The virB genes coding type IV secretion system are necessary for the intracellular survival and replication of Brucella spp. In this study, extracellular proteins from B. abortus 2308 (wild type, WT) and its isogenic virB10 polar mutant were compared. Culture supernatants harvested in the early stationary phase were concentrated and subjected to 2D electrophoresis. Spots present in the WT strain but absent in the virB10 mutant (differential spots) were considered extracellular proteins released in a virB-related manner, and were identified by MALDI-TOF analysis and matching with Brucella genomes. Among the 11 differential proteins identified, DnaK chaperone (Hsp70), choloylglycine hydrolase (CGH) and a peptidyl-prolyl cis-trans isomerase (PPIase) were chosen for further investigation because of their homology with extracellular and/or virulence factors from other bacteria. The three proteins were obtained in recombinant form and specific monoclonal antibodies (mAbs) were prepared. By Western blot with these mAbs, the three proteins were detected in supernatants from the WT but not in those from the virB10 polar mutant or from strains carrying non-polar mutations in virB10 or virB11 genes. These results suggest that the expression of virB genes affects the extracellular release of DnaK, PPIase and CGH, and possibly other proteins from B. abortus.

Dipentamethylene thiuram monosulfide is a novel inhibitor of Pin1

Pin1 is involved in eukaryotic cell proliferation by changing the structure and function of phosphorylated proteins. PiB, the Pin1 specific inhibitor, blocks cancer cell proliferation. However, low solubility of PiB in DMSO has limited studies of its effectiveness. We screened for additional Pin1 inhibitors and identified the DMSO-soluble compound dipentamethylene thiuram monosulfide (DTM) that inhibits Pin1 activity with an EC50 value of 4.1 μM. Molecular modeling and enzyme kinetic analysis indicated that DTM competitively inhibits Pin1 activity, with a K i value of 0.05 μM. The K D value of DTM with Pin1 was determined to be 0.06 μM by SPR technology. Moreover, DTM specifically inhibited peptidyl–prolyl cis/ trans isomerase activity in HeLa cells. FACS analysis showed that DTM induced G0 arrest of the HCT116 cells. Our results suggest that DTM has the potential to guide the development of novel antifungal and/or anticancer drugs.

A peptidyl–prolyl isomerase, FKBP12, accumulates in Alzheimer neurofibrillary tangles

We investigated a possible role in Alzheimer's disease (AD) for FKBP12, a peptidyl–prolyl cis–trans isomerase known to be important in protein assembly, folding and transportation by using Western blotting and microscopic analyses in postmortem brain tissues from elderly controls and the patients with AD. FKBP12 was enriched and localized to neuronal cell bodies and neurites in control brains. Intense immunoreactivity was found in large neurons such as pyramidal cells. Many FKBP12 positive granules were located in the cytoplasm and the proximal portion of dendrites and axons, and in the nuclei. By contrast, the expression of FKBP12 in AD brains was lower than in control brains. Furthermore, numerous intracellular neurofibrillary tangles (NFTs) were stained for FKBP12 in the hippocampal CA1 subfield, subiculum, entorhinal cortex and angular gyrus. Neuritic pathology such as neuropil threads and dystrophic neurites (DNs) within senile plaques (SPs) and some reactive astrocytes were also immunolabeled for FKBP12 in AD. Double immunofluorescence staining showed dual labeling of intracellular NFTs for FKBP12 and tau. Similar results were obtained in reactive astrocytes for the combination of FKBP12 and glial fibrillary acidic protein (GFAP). Labeling for FKBP12 was dense in axons stained for highly phosphorylated neurofilament protein. Thus our results suggest that FKBP12 may be involved in neuronal or astrocytic cytoskeletal organization and in the abnormal metabolism of tau protein in AD damaged neurons.

Structural rearrangements and the unfolding mechanism of a Trigger Factor mutant studied by multiple structural probes

Trigger Factor (TF) is a three-domain chaperone which catalyzes nascent peptide folding and harbors peptidyl–prolyl cis–trans isomerase activity. The multi-domain structure of TF makes it an interesting and challenging candidate for studies of the structural properties and functional behavior of individual domains or combined domain constructs. Here we constructed a TF mutant, NC, combining the N- and C-domains that are responsible for TF's chaperone function, and compared structural changes and unfolding characteristics of NC with wild-type TF by monitoring fluorescence spectra, far-UV CD, chemical crosslinking, DSC and binding with hydrophobic probes (ANS or bis-ANS). The results showed that the NC construct, like intact TF, could bind to hydrophobic probes, form dimers in solution, and showed a similar 3-state guanidine-induced unfolding profile. However, the NC fragment showed reduced stability towards both guanidine unfolding and thermal denaturation, suggesting that the presence of the M-domain of TF contributes to the stability of the intact TF structure.

Differential Responses of the Backbone and Side-Chain Conformational Dynamics in FKBP12 upon Binding the Transition-State Analog FK506: Implications for Transition-State Stabilization and Target Protein Recognition

FKBP12 serves a dual role as a peptidyl-prolyl cis-trans isomerase and as a modulator of several cell signaling pathways. The macrolide FK506 is a transition-state analog of the catalyzed reaction and displaces FKBP12 from its natural target proteins. We compared the conformational exchange dynamics of the backbone and methyl-bearing side chains of FKBP12 in the free and FK506-bound states using NMR relaxation-dispersion experiments. Our results show that the free enzyme exchanges between the ground state and an excited state that resembles the ligand-bound state or Michaelis complex. In FK506-bound FKBP12, the backbone is confined to a single conformation, while conformational exchange prevails for many methyl groups. The residual side-chain dynamics in the transition-state analog-bound state suggests that the transition-state ensemble involves multiple conformations, a finding that challenges the long-standing concept of conformational restriction in the transition-state complex. Furthermore, exchange between alternative conformations is observed in the bound state for an extended network of methyl groups that includes locations remote from the active site. Several of these locations are known to be important for interactions with cellular target proteins, including calcineurin and the ryanodine receptor, suggesting that the conformational heterogeneity might play a role in the promiscuous binding of FKBP12 to different targets.

Identification and analysis of genes encoding a novel ER-localised Cyclophilin B in wheat potentially involved in storage protein folding

The storage protein deposition processes in cereal endosperm follow a strict script involving protein folding and refolding, assortment and protein body deposition by two distinct pathways. The endoplasmic reticulum-localised enzymes called peptidyl prolyl cis– trans isomerases (PPIases) are expected to play crucial ‘foldase’ and chaperone roles in the deposition of wheat prolamins due to their proline-rich nature. The storage protein quality of wheat is important for nutritional and food technological purposes; however, little information exists on genetic control of protein sorting and deposition processes. In this study, CypB genes encoding an ER-localised cyclophilin (a PPIase) have been characterised from common wheat ( Triticum aestivum L. AABBDD) and its progenitors for the first time. The full-length genes contain seven exons and six introns, the exons being significantly more conserved. The putative CypB protein contains all PPIase signature sites including the putative active site and cyclosporin-binding residues, but an atypical ER localisation signal. Using the sequence variations, the common wheat genes were localized to chromosomes 7AL, 7BL and 7DL. The promoters of wheat genes were identified by inverse-PCR and show a number of potential tissue specific regulatory elements. The wheat genes are similar in structure to rice CypB and the putative proteins are 83% identical. Further, certain QTLs related to protein quality occur at the rice and wheat CypB loci. The results support this enzyme being a strong candidate for regulating storage protein quality in wheat.

Functional fusion mutant of Candida antarctica lipase B (CalB) expressed in Escherichia coli

Candida antarctica lipase B (CalB) was functionally expressed in the cytoplasm of Escherichia coli Origami(DE3) with the N-terminus fusion of E. coli endogenous proteins. The previously-identified stress responsive proteins through comparative proteome analyses such as malate dehydrogenase (Mdh), spermidine/putrescine-binding periplasmic protein (PotD), and FKBP-type peptidyl–prolyl cis–trans isomerase (PPIases) (SlyD) dramatically increased the solubility of CalB in E. coli cytoplasm when used as N-terminus fusion partners. We demonstrated that Mdh, PotD, and SlyD were powerful solubility enhancers that presumably facilitated the protein folding of CalB. Moreover, among the various fusion mutants, Mdh-CalB showed the highest hydrolytic activity and was as biologically active as standard CalB. Similarly to the previous report, the electrophoretic properties of CalB indicate that CalB seems to form dimer-based oligomer structures. We evaluated the structural compatibility between the fusion partner protein and CalB, which seems to be of crucial importance upon the bioactive dimer formation of CalB and might affect the substrate accessibility to the enzyme active site, thereby determining the biological activities of the fusion mutants.

Mechanistic Insight into the Role of Transition-State Stabilization in Cyclophilin A

Peptidyl prolyl cis−trans isomerases (PPIases) are ubiquitous enzymes in biology that catalyze the cis−trans isomerization of the proline imide peptide bond in many cell signaling pathways. The local change of the isomeric state of the prolyl peptide bond acts as a switching mechanism in altering the conformation of proteins. A complete understanding of the mechanism of PPIases is still lacking, and current experimental techniques have not been able to provide a detailed atomistic picture. Here we have carried out several accelerated molecular dynamics simulations with explicit solvent, and we have provided a detailed description of cis−trans isomerization of the free and cyclophilin A-catalyzed process. We show that the catalytic mechanism of cyclophilin is due mainly to the stabilization and preferential binding of the transition state that is achieved by a favorable hydrogen bond interaction with a backbone NH group. We also show that the substrate in the transition state interacts more favorably with the enzyme than the cis isomer, which in turn interacts more favorably than the trans isomer. The stability of the enzyme−substrate complex is directly correlated with the interaction the substrate makes with a highly conserved arginine residue. Finally, we show that catalysis is achieved through the rotation of the carbonyl oxygen on the N-terminal of the prolyl peptide bond in a predominately unidirectional fashion.

Involvement of cyclophilin D in mitochondrial permeability transition induction in intact cells

The mitochondrial permeability transition (MPT) is involved in both Ca 2+ signaling and cell death. The present study aimed to clarify the involvement of cyclophilin D, a peptidyl prolyl cis– trans isomerase (PPIase), in MPT induction in intact cells. To achieve this, we used C6 cells overexpressing wild-type or PPIase-deficient cyclophilin D, and measured the inner mitochondrial membrane permeability to calcein, a 623-Da hydrophilic fluorescent molecule, to evaluate MPT induction. In vector control cells, the percentage of MPT induction by ionomycin increased as the Ca 2+ concentration in the extracellular medium increased. This result indicates that the present method is valid for numerical evaluation of MPT induction. In C6 cells expressing the PPIase-deficient mutant, the percentage of MPT induction was significantly decreased compared with wild-type CypD-overexpressing cells or vector control cells. These results suggest that cyclophilin D is involved in MPT induction by Ca 2+ in intact cells.

The Dual Histidine Motif in the Active Site of Pin1 Has a Structural Rather than Catalytic Role

The catalytic domain of the peptidyl-prolyl cis/ trans isomerase Pin1 is a member of the FKBP superfold family. Within its active site are two highly conserved histidine residues, H59 and H157. Despite their sequence conservation in parvulin PPIase domains, the role of these histidine residues remains unclear. Our previous work (Behrsin et al. (2007) J. Mol. Biol. 365, 1143- 1162.) was consistent with a model where one or both histidines had critical roles in a hydrogen bonding network in the active site. Here, we test this model by looking at the effect of mutations to H59 and H157 on Pin1 function, activity, and protein stability. Using a yeast complementation assay, we show that both H59 and H157 can be mutated to non-hydrogen bonding residues and still support viability. Surprisingly, a nonfunctional H59L mutation can be rescued by a mutation of H157, to leucine. This double mutation (H59L/H157L) also had about 5-fold greater isomerase activity than the H59L mutation with a phosphorylated substrate. Structural analyses suggest that rescue of function and activity results from partial rescue of protein stability. Our findings indicate that H59 and H157 are not required for hydrogen bonding within the active site, and in contrast to the active site C113, they do not participate directly in catalysis. Instead, we suggest these histidines play a key role in domain structure or stability.

Identification of all FK506-binding proteins from Neurospora crassa

Immunophilins are intracellular receptors of immunosuppressive drugs, carrying peptidyl-prolyl cis–trans isomerase activity, with a general role in protein folding but also involved in specific regulatory mechanisms. Four immunophilins of the FKBP-type (FK506-binding proteins) were identified in the genome of Neurospora crassa. Previously, FKBP22 has been located in the endoplasmic reticulum as part of chaperone/folding complexes and FKBP13 has been found to have a dual location in the cytoplasm and mitochondria. FKBP11 is apparently located exclusively in the cytoplasm. It is not expressed during vegetative development of the fungus although its expression can be induced with calcium and during sexual development. Overexpression of the respective gene appears to confer a growth advantage to the fungus in media containing some divalent ions. FKBP50 is a nuclear protein and its genetic inactivation leads to a temperature-sensitive phenotype. None of these proteins is, alone or in combination, essential for N. crassa, as demonstrated by the isolation of a mutant strain lacking all four FKBPs.

Cyclophilin-40 has a cellular role in the aryl hydrocarbon receptor signaling

Cyclophilin-40 (CyP40) promotes the formation of the gel shift complex that contains the aryl hydrocarbon receptor (AhR), AhR nuclear translocator (Arnt) and dioxin response element (DRE) using baculovirus expressed proteins. Here we reported that CyP40 plays a role in the AhR signaling. When the CyP40 content in MCF-7 cells is reduced, up-regulation of cyp1a1 and cyp1b1 by 3-methylchloranthrene (3MC) is also reduced, suggesting that CyP40 is essential for maximal AhR function. The CyP40 region containing amino acids 186–215, but not the peptidyl-prolyl cis– trans isomerase and tetratricopeptide repeat domains, is essential for forming the AhR/Arnt/DRE complex. CyP40 is found in the cell nucleus after 3MC treatment and appears to promote the DRE binding form of the AhR/Arnt heterodimer.

Biochemical function of typical and variant Arabidopsis thaliana U-box E3 ubiquitin-protein ligases

The variance of the U-box domain in 64 Arabidopsis thaliana (thale cress) E3s (ubiquitin-protein ligases) was used to examine the interactions between E3s and E2s (ubiquitin-conjugating enzymes). E2s and E3s are components of the ubiquitin protein degradation pathway. Seven U-box proteins were analysed for their ability to ubiquitinate proteins in vitro in co-operation with different E2s. All U-box domains exhibited ubiquitination activity and interacted productively with UBC4/5-type E2s. Three and four of the U-box domains mediated ubiquitin addition in the presence of UBC13 and UBC7 E2s respectively, but no productive interaction was observed with the UBC15 E2 tested. The activity of AtPUB54 [Arabidopsis thaliana (thale cress) plant U-box 54 protein] was dependent on Trp(266) in the E2-binding cleft, and the E2 selectivity was changed by substitution of this position. The function of the distant U-box protein, AtPUB49, representing a large family of eukaryotic proteins containing a U-box linked to a cyclophilin-like peptidyl-prolyl cis-trans isomerase domain, was characterized biochemically. AtPUB49 functioned both as a prolyl isomerase and a chaperone by catalysing cis-trans isomerization of peptidyl-prolyl bonds and dissolving protein aggregates. In conclusion, both typical and atypical Arabidopsis U-box proteins were active E3s. The overlap in the E3/E2 selectivity suggests that in vivo specificity is not determined only by the E3-E2 interactions, but also by other parameters, e.g. co-existence or interactions with additional domains. The biochemical functions of AtPUB49 suggest that the protein can be involved in folding or degradation of protein substrates. Similar functions can also be retained within a protein complex with separate chaperone and U-box proteins.

The Arabidopsis X-prolyl protease/cis trans isomerase APP1 regulates auxin signal transduction via interactions with AUX/IAA proteins

The Arabidopsis X-prolyl protease/cis trans isomerase APP1 regulates auxin signal transduction via interactions with AUX/IAA proteins

Spliceosomal immunophilins

The spliceosome is a dynamic, macromolecular complex, which removes non-protein-coding introns from pre-mRNA to form mature mRNA in a process known as splicing. This ribonucleoprotein assembly is comprised of five uridine-rich small nuclear RNAs (snRNAs) as well as over 300 proteins. In humans, several of the known proteinaceous splicing factors are members of the immunophilin superfamily. Immunophilins are peptidyl-prolyl cis– trans isomerases that catalyze the conversion of proteins from cis to trans at Xaa-Pro bonds. Our review of the data indicates that some members of this protein family are activators of spliceosomal proteins by way of folding and transport.

FKBP36 Forms Complexes with Clathrin and Hsp72 in Spermatocytes

The testes-specific peptidyl-prolyl cis/ trans isomerase FKBP36 plays a crucial role in male meiosis. Here we show that the catalytic domain of FKBP36 binds to clathrin heavy chain (CHC) of clathrin. Despite wild-type FKBP36 not displaying PPIase activity, the introduction of the R81L substitution resulted in catalysis of prolyl isomerization, which is comparable to the regulated activity of FKBP38. Furthermore, the TPR domain of FKBP36 interacts with Hsp72. In fact, FKBP36 preferentially binds to Hsp72 among the members of the Hsp70 family and is thus the first TPR-containing protein which discriminates between Hsp70 proteins. The clathrin-FKBP36-Hsp72 complexes resulting from both identified interactions are bound to the matrices of clathrin-coated vesicles in spermatocytes, which indicates a possible role of FKBP36 and Hsp72 in the disassembly of clathrin coats.

Bioactivity of Folding Intermediates Studied by the Recovery of Enzymatic Activity during Refolding

The peptide bond preceding proline residues realizes a cis/ trans conformational switch with high switching resistance in native proteins and folding intermediates. Therefore, individual isomers have the potential to differ in bioactivity. However, information about isomer-specific bioactivities is difficult to obtain because of the risk of affecting isomeric distribution by bioactivity assay components. Here we present an approach that allows for the measurement of the recovery of enzymatic activities of wild-type RNase T 1 and RNase T 1 variants during refolding under conditions where the population of enzyme–substrate or enzyme–product complexes is negligible. Recovery of enzymatic activity was continuously monitored within the visible range of the spectrum by addition of a fluorescence-labeled nucleotide substrate to the refolding sample. We found that a nonnative trans conformation at Pro39 renders the RNase T 1 almost completely inactive. A folding intermediate having a nonnative trans conformation at Pro55 shows about 46% of the enzymatic activity referred to the native state. Pro55, in contrast to the active site located Pro39, is situated in a solvent-exposed loop region remote from active-site residues. In both cases, peptidyl prolyl cis/ trans isomerases accelerate the regain of nucleolytic activity. Our findings show that even if there is a considerable distance between the site of isomerization and the active site, conformational control of the bioactivity of proteins is likely to occur, and that the surface location of prolyl bonds suffices for the control of buried active sites mediated by peptidyl prolyl cis/ trans isomerases.

The hippocampal protein machinery varies over the estrous cycle

Information about estrous cycle (EC) and sex-dependent protein levels is limited. Cognitive functions vary over the EC and the aim of this study was to investigate rat protein fluctuations in the hippocampus, the main cognitive brain area for learning and memory, in the individual phases of the EC and in males and indeed protein fluctuations may reflect functional variation over the EC. Sprague-Dawley rats were used in the studies and estrous phases were determined. Hippocampi were taken, proteins extracted, run on 2-DE, and identified using MALDI-TOF/TOF and nano-LC-ESI-MS/MS; protein levels were quantified using Proteomweaver software. Levels of protein synthetic machinery components transcriptional activator protein PUR(α,β), elongation factor 2, heterogeneous nuclear ribonucleoprotein K, chaperones 78 kDa glucose-regulated protein, heat shock cognate 71 kDa protein, Hsp 105, stress-70 protein, peptidyl-prolyl cis-trans isomerase A, prefoldin subunit 2, T-complex protein 1 subunit alpha and subunit delta, and degradation principle proteasome subunit alpha type 1 and ubiquitin carboxyl-terminal hydrolase isozyme L1, were different between sex and phase of the EC. We suggest that differences in the protein synthetic, chaperoning, and degradation machinery indicate different function in the individual EC phases. Results herein are relevant for further design of studies in the hippocampus at the protein level and interpretation of previous studies because EC phases will have to be respected and taken into account.

Cyclophilin A differentially activates monocytes and endothelial cells: Role of purity, activity, and endotoxin contamination in commercial preparations

BackgroundCyclophilin A (CyPA) is a cytoplasmic protein secreted under inflammatory conditions. Extracellular CyPA is detected in atherosclerotic plaques and has been observed to activate endothelial cells as well as monocytes. Methods and resultsCommercially available recombinant CyPA-induced expression of tissue factor (TF) and vascular cell adhesion molecule-1 (VCAM-1) in human aortic endothelial cells (HAEC). However, CyPA from commercial sources contained lipopolysaccharide at concentrations up to 18.9ng/ml; moreover, it exhibited low purity as determined by protein spectrum analysis and low activity as assessed by peptidyl prolyl cis–trans isomerase (PPIase) assay. An in-house preparation of pure, active, and uncontaminated CyPA failed to induce endothelial TF or VCAM-1 expression; moreover, it was not chemotactic for HAEC. In contrast, such CyPA exhibited potent chemotactic activity on monocytic THP-1 cells, with a maximal effect on migration occurring at a concentration of 5.5×10−9mol/l. Pretreatment of CyPA with cyclosporine A prevented its effect on THP-1 cell migration; similarly, PPIase-deficient mutant CyPA protein did not induce migration of these cells. In-house prepared CyPA induced the release of Il-6, but not TNF-α, from THP-1 cells. ConclusionsCommercially available CyPA exhibits low purity and activity and may be contaminated by endotoxin. Pure, active, and uncontaminated CyPA does not induce endothelial TF or VCAM-1 expression; instead, it acts as a potent monocyte chemoattractant and induces monocyte Il-6 release, implying a role for extracellular CyPA in the pathogenesis of atherosclerosis via activation of monocytes rather than endothelial cells.