Microbial cyclophilins: specialized functions in virulence and beyond

Cyclophilins belong to the superfamily of peptidyl-prolyl cis/trans isomerases (PPIases, EC: 5.2.1.8), the enzymes that catalyze the cis/trans isomerization of peptidyl-prolyl peptide bonds in unfolded and partially folded polypeptide chains and native state proteins. Cyclophilins have been extensively studied, since they are involved in multiple cellular processes related to human pathologies, such as neurodegenerative disorders, infectious diseases, and cancer. However, the presence of cyclophilins in all domains of life indicates a broader biological importance. In this mini-review, we summarize current advances in the study of microbial cyclophilins. Apart from their anticipated role in protein folding and chaperoning, cyclophilins are involved in several other biological processes, such as cellular signal transduction, adaptation to stress, control of pathogens virulence, and modulation of host immune response. Since many existing family members do not have well-defined functions and novel ones are being characterized, the requirement for further studies on their biological role and molecular mechanism of action is apparent.     

Characterization of Peptidyl-Prolyl Cis-Trans Isomerase- and Calmodulin-Binding Activity of a Cytosolic Arabidopsis thaliana Cyclophilin AtCyp19-3

Cyclophilins, which bind to immunosuppressant cyclosporin A (CsA), are ubiquitous proteins and constitute a multigene family in higher organisms. Several members of this family are reported to catalyze cis-trans isomerisation of the peptidyl-prolyl bond, which is a rate limiting step in protein folding. The physiological role of these proteins in plants, with few exceptions, is still a matter of speculation. Although Arabidopsis genome is predicted to contain 35 cyclophilin genes, biochemical characterization, imperative for understanding their cellular function(s), has been carried only for few of the members. The present study reports the biochemical characterization of an Arabidopsis cyclophilin, AtCyp19-3, which demonstrated that this protein is enzymatically active and possesses peptidyl-prolyl cis-trans isomerase (PPIase) activity that is specifically inhibited by CsA with an inhibition constant (K_i) of 18.75 nM. The PPIase activity of AtCyp19-3 was also sensitive to Cu²⁺, which covalently reacts with the sulfhydryl groups, implying redox regulation. Further, using calmodulin (CaM) gel overlay assays it was demonstrated that in vitro interaction of AtCyp19-3 with CaM is Ca²⁺-dependent, and CaM-binding domain is localized to 35–70 amino acid residues in the N-terminus. Bimolecular fluorescence complementation assays showed that AtCyp19-3 interacts with CaM in vivo also, thus, validating the in vitro observations. However, the PPIase activity of the Arabidopsis cyclophilin was not affected by CaM. The implications of these findings are discussed in the context of Ca²⁺ signaling and cyclophilin activity in Arabidopsis.     

Cyclophilin inhibitors as antiviral agents

Cyclophilins (Cyps) are ubiquitous proteins that effect the cis–trans isomerization of Pro amide bonds, and are thus crucial to protein folding. CypA is the most prevalent of the ～19 human Cyps, and plays a crucial role in viral infectivity, most notably for HIV-1 and HCV. Cyclophilins have been shown to play key roles in effective replication of a number of viruses from different families. A drug template for CypA inhibition is cyclosporine A (CsA), a cyclic undecapeptide that simultaneously binds to both CypA and the Ca²⁺-dependent phosphatase calcineurin (CN), and can attenuate immune responses. Synthetic modifications of the CsA scaffold allows for selective binding to CypA and CN separately, thus providing access to novel, non-immunosuppressive antiviral agents.     

CNS1 Encodes an Essential p60/Sti1 Homolog in Saccharomyces cerevisiae That Suppresses Cyclophilin 40 Mutations and Interacts with Hsp90

Cyclophilins are cis-trans-peptidyl-prolyl isomerases that bind to and are inhibited by the immunosuppressant cyclosporin A (CsA). The toxic effects of CsA are mediated by the 18-kDa cyclophilin A protein. A larger cyclophilin of 40 kDa, cyclophilin 40, is a component of Hsp90-steroid receptor complexes and contains two domains, an amino-terminal prolyl isomerase domain and a carboxy-terminal tetratricopeptide repeat (TPR) domain. There are two cyclophilin 40 homologs in the yeast Saccharomyces cerevisiae, encoded by the CPR6 and CPR7 genes. Yeast strains lacking the Cpr7 enzyme are viable but exhibit a slow-growth phenotype. In addition, we show here that cpr7 mutant strains are hypersensitive to the Hsp90 inhibitor geldanamycin. When overexpressed, the TPR domain of Cpr7 alone complements both cpr7 mutant phenotypes, while overexpression of the cyclophilin domain of Cpr7, full-length Cpr6, or human cyclophilin 40 does not. The open reading frame YBR155w, which has moderate identity to the yeast p60 homolog STI1, was isolated as a high-copy-number suppressor of the cpr7 slow-growth phenotype. We show that this Sti1 homolog Cns1 (cyclophilin seven suppressor) is constitutively expressed, essential, and found in protein complexes with both yeast Hsp90 and Cpr7 but not with Cpr6. Cyclosporin A inhibited Cpr7 interactions with Cns1 but not with Hsp90. In summary, our findings identify a novel component of the Hsp90 chaperone complex that shares function with cyclophilin 40 and provide evidence that there are functional differences between two conserved sets of Hsp90 binding proteins in yeast.     

Multistage affinity cross-flow filtration: process optimization

The recovery yield (REC) and productivity (PRD) are used as objective functions to optimize the multistage affinity cross-flow filtration (mACFF) process. The effects of the operating conditions such as feed loading volume (Q _L ⁺), total protein concentration and target protein purity in the feeding broth are analyzed. For higher affinity system or by a mACFF process with larger number of stages as well as more macroligand loading, there is a critical value of Q _L ⁺ below which the REC keeps constant and maximal. This maximal value of REC is affected by the stage number as well as macroligand loading of the mACFF process and the affinity system (i.e., the binding constant of the target protein to its macroligand), but independent of the feeding broth properties (i.e., total protein concentration and target protein purity) and membrane permeability. An optimum of Q _L ⁺ exists to give a maximum of PRD. The optimal Q _L ⁺ is somewhat larger than the critical Q _L ⁺ value below which REC keeps constant. The maximum of PRD is raised by increasing the stage number and macroligand loading of the mACFF process, affinity binding constant, and total protein concentration as well as target protein purity in the feeding broth, but reduced by increasing the membrane rejection coefficient (R). However, it is encouraging that the decrease of the maximal PRD is less significant when R is less than 0.5. Therefore, if it is not possible to find a membrane that is completely permeable to proteins and at the same time completely impermeable to the macroligand, a membrane with R less than 0.5 may be selected to obtain a larger PRD. The results obtained in this work give further predictive understanding of the mACFF technique, and will be useful to the process design.     

Direct assay for O6-methylguanine-acceptor protein in cell extracts

A direct in vitro assay for O⁶-methylguanine-acceptor protein in cell extracts that measures the transfer of radioactivity from labeled O⁶-methylguanine (O⁶MeGua) adducts in an exogenous DNA substrate to protein is described. The protein-bound radioactivity is released and separated from that remaining in the DNA by sequential digestion with protease K and aminopeptidase M, and appears in the alcohol-soluble fraction of the digest. Data obtained by the direct assay are similar to those obtained by an indirect assay that measures the amount of O⁶MeGua-acceptor protein as the loss of O⁶MeGua from the DNA. In addition to its accuracy, the direct assay is also simple and can measure the amount of O⁶MeGua-acceptor activity in cell extracts prepared from as few as 0.5–1.0 × 10⁶ mammalian cells.     

Molecular mechanism for dominance of a mutant allele of an ATP-dependent protease

We have demonstrated that the lon⁺ (capR⁺) ATP-hydrolysis-dependent protease is inhibited by the addition of an enzymatically inactive mutant form of the protein (capR9 protein). The enzymatic activity of the capR⁺-capR9 protein mixture is also more stable to heat than the capR⁺ protein alone indicating that hybrid molecules are formed. Independent measurements demonstrated that the lon⁺ ATP-hydrolysis-dependent protease is a tetramer of identical 94 × 10₃ molecular weight monomers. The pattern of defective subunit (capR9) inhibition indicates only tetramers containing three capR9 monomers and one capR⁺ monomer are inactive, i.e. two or more capR⁺ monomers are required for a tetramer to act as an ATP-hydrolysis-dependent protease. Two molecular mechanisms to explain the dominance of a mutant allele, termed anticomplementation, are considered.     

Proteinbiosynthesen in dormanten und nachgereiften embryonen und samen von Agrostemma githago

Seed germination of Agrostemma githago is prevented by inhibitors of protein and RNA synthesis. Thus protein as well as RNA synthesis are essential prerequisites for germination. Early protein synthesis of Agrostemnia embryos can be completely inhibited by cycloheximide and cordycepin. During the aging of seeds there is a considerable decrease in germination capacity and protein synthesis. In dormant and afterripened embryos of Agrostemma githago¹⁴C-leucine and ¹⁴C-uracil are incorporated in protein and RNA respectively with nearly the same intensity, whereas RNA and protein synthesis of dormant seeds and embryos starts earlier than in those subjected to afterripening. ³H-uracil-labelled RNA from dormant and afterripened embryos are able to hybridize on oligo-dT-cellulose to the same extent. There is a similarity in the protein pattern of dormant and afterripened embryos revealed by electrophoresis in polyacrylamide gels of double-labelled proteins. According to these results dormancy of Agrostemma githago is not caused by a general but by a specific metabolic block.     

The role of copper binding in the conformation of stellacyanin

The binding of Cu²⁺ to apostellacyanin occurs in two steps. The first step consists of a fast equilibrium reaction involving binding of copper to the protein in a non-native, though specific way, as shown by electron paramagnetic resonance measurements. All the spectroscopic properties of native stellacyanin are recovered in a slower monomolecular process (k = 7.5 × 10^?3 sec^?1 at 25 °C) characterized by high activation energy (ΔH_a = 22 kcal mole^?1) and low activation entropy (ΔS_a = 3.0 cal deg^?1 mole^?1). The second step parallels a conformational change of the copper-bound protein molecule. A large difference of the tyrosyl residues pKs is found between holo- and apostellacyanin. In the latter the tyrosyl residues appear to be more exposed to solvent perturbations. Ammonia or monovalent anions such as N₃^?, SCN^?, and Cl^? have a catalytic effect on the second step of the reaction, roughly proportional to their first binding constant to aqueous copper. It is suggested that they may compete for a non-native bond of the copper to the protein, thus rendering the conformational change easier.The effect of Ag³ and Hg²⁺ on the recombination reaction with copper is discussed in terms of conformation of the metal-bound protein.     

1H, 13C and 15N backbone and side-chain resonance assignments of reduced CcmG from Escherichia coli

CcmG is a periplasmic, membrane-anchored protein widely distributed in a variety of species. In Escherichia coli, the CcmG protein always acts as a weak reductant in the electron transport chain during cytochrome c maturation (Ccm). Here we report ¹H, ¹⁵N and ¹³C backbone and side-chain resonance assignments of the reduced CcmG protein (residues 19–185, renumbered as 1–167) from E. coli. This work lays the essential basis for the further structural and functional analysis of reduced CcmG.     

NMR resonance assignment of DnaE intein from Nostoc punctiforme

DnaE intein from Nostoc punctiforme (Npu) is one of naturally occurring split inteins, which has robust protein splicing activity. Highly efficient trans-splicing activity of NpuDnaE intein could widen various biotechnological applications. However, structural basis of the efficient protein splicing activity is poorly understood. As a first step toward better understanding of protein trans-splicing mechanism, we present the backbone and side-chain resonance assignments of a single chain variant NpuDnaE intein as determined by triple resonance experiments with [¹³C,¹⁵N]-labeled protein.     

Purification of a protein from Nitellopsis obtusa cells and its involvement in the regulation of potential-dependent Ca2+ channels

A protein was isolated from the thermostable protein fraction of N. obtusa cells and purified by hydrophobic chromatography on phenyl-Sepharose and affinity chromatography on melittin-Sepharose. In 15% polyacrylamide gel, the protein has an electrophoretic mobility corresponding to M_r 17,000 in the presence of 1 mM Ca²⁺ and M_r no higher than 19,000 in the presence of 1 mM EGTA. Introduction of the protein isolated to a perfused N. obtusa cell affects the electric parameters of the plasmalemma Ca²⁺ channels. This influence shows up as a change in I_Ca²⁺, as well as an activation of the electrogenous processes in the plasmalemma. The protein produces restoration of I_Ca²⁺ in the Ca²⁺ channels blocked by chlorpromazine. Possible mechanisms of involvement of this protein in regulation of the functional state of potential-dependent Ca²⁺ channels of N. obtusa plasmalemma are assumed.     

Ovate family protein 1 as a plant Ku70 interacting protein involving in DNA double-strand break repair

The Ku heterodimer, a DNA repair protein complex consisting of 70- and 80-kDa subunits, is involved in the non-homologous end-joining (NHEJ) pathway. Plants are thought to use the NHEJ pathway primarily for the repair of DNA double-strand breaks (DSBs). The Ku70/80 protein has been identified in many plants and been shown to possess several similar functions to its counter protein complex in mammals. In the present study, ovate family protein 1 (AtOFP1) was demonstrated to be a plant Ku-interacting protein by yeast two-hybrid screening and the GST pull-down assay. Truncation analysis revealed that the C-terminal domain of AtKu70 contains interacting sites for AtOFP1. The electrophoretic mobility shift assay (EMSA) indicated that AtOFP1 is also a DNA binding protein with its binding domain at the N-terminus. In 3-week-old seedlings, expression of the AtOFP1 gene increased after exposure to DNA-damaging agents (such as methyl methanesulfonate (MMS) and menadione) in a time dependent manner. Seedlings lacking the AtOFP1 protein were more sensitive to MMS and menadione as compared with wild-type. Furthermore, similar to AtKu70 ^?/? and AtKu80 ^?/?, the AtOFP1 ^?/? mutant showed relatively lower NHEJ activity in vivo. Taken together, these results suggest that AtOFP1 may play a role in DNA repair through the NHEJ pathway accompanying with the AtKu protein.     

Synthesis, transport and localization of a nuclear coded 22-kd heat-shock protein in the chloroplast membranes of peas and Chlamydomonas reinhardi

The synthesis, transport and localization of a nuclear coded 22-kd heat-shock protein (HSP) in the chloroplast membranes was studied in pea plants and Chlamydomonas reinhardi. HSPs were detected in both systems by in vivo labeling and in vitro translation of poly(A)⁺RNA, using the wheat-germ and reticulocyte lysate systems. Heat-shock treatment of pea plants for 2 h at 42-45°C induces the expression of ˜10 nuclear coded proteins, among which several (18 kd, 19 kd, 22 kd) are predominant. A 22-kd protein is synthesized as a 26-kd precursor protein and is localized in a chloroplast membrane fraction in vivo. Following post-translational transport into intact chloroplasts in vitro of the 26-kd precursor, the protein is processed but the resulting 22-kd mature protein is localized in the chloroplast stroma. If, however, the in vitro transport is carried out with chloroplasts from heat-shocked plants, the 22-kd protein is preferentially transported to the chloroplast membrane fraction. In C. reinhardi the synthesis of poly(A)⁺RNAs coding for several HSPs is progressively and sequentially induced when raising the temperature for 1.5 h from 36°C to 42°C, while that of several preexisting RNAs is reduced. Various pre-existing poly(A)⁺RNAs endure in the cells at 42°C up to 5 h but are no longer translated in vivo, whereas some poly(A)⁻RNAs persist and are translated. As in pea, a poly(A)⁺RNA coded 22-kd HSP is localized in the chloroplast membranes in vivo, although it is translated as a 22-kd protein in vitro. The in vitro translated protein is not transported in isolated pea chloroplast which, however, processes and transports other nuclear coded chloroplast proteins of Chlamydomonas. The poly(A)⁺RNA coding for the 22-kd HSP appears after 1 h at 36°C. Its synthesis increases with the temperature of incubation up to 42°C, although it decreases after ˜2 h of heat treatment and the already synthesized RNA is rapidly degraded. The degradation is faster upon return of the cells to 26°C. None of the heat-induced proteins is identical to the light-inducible proteins of the chloroplast membranes.     

The armadillo repeat domain of Apc suppresses intestinal tumorigenesis

The adenomatous polyposis coli (APC) gene is known to act as a tumor suppressor gene in both sporadic and hereditary colorectal cancer by negatively regulating WNT signaling. Familial adenomatous polyposis (FAP) patients develop intestinal polyps due to the presence of a single germline mutation in APC. The severity of the FAP phenotype is a function of the position of the APC mutation, indicating a complex role for APC that extends beyond the canonical WNT pathway. APC encodes a large protein with multiple functional domains, including an armadillo repeat domain that has been linked to protein–protein interactions. To determine the effect of the armadillo repeat domain on intestinal tumorigenesis, we generated a congenic mouse line (Apc ^Δ242 ) carrying a gene trap cassette between exons 7 and 8 of the murine Apc gene. Apc ^Δ242/+ mice express a truncated Apc product lacking the armadillo repeat domain as part of a fusion protein with β-geo. Expression of the fusion product was confirmed by X-gal staining, ensuring that Apc ^Δ242 is not a null allele. In contrast, Apc ^Min/+ mice produce a truncated Apc product that contains an intact armadillo repeat domain. On the C57BL/6J background, Apc ^Δ242/+ mice develop more polyps than do Apc ^Min/+ mice along the entire length of the small intestine; however, polyps were significantly smaller in Apc ^Δ242/+ mice. In addition, polyp multiplicity in Apc ^Δ242/+ mice is affected by polymorphisms between inbred strains. These data suggest that the armadillo repeat domain of the Apc protein suppresses tumor initiation in the murine intestine while also promoting tumor growth.