ABSTRACT: Several single-domain prokaryotic and eukaryotic cyclophilins have been identified as also being unspecific nucleases with a role in DNA degradation during the lytic processes that accompany bacterial cell death and eukaryotic apoptosis. Evidence is provided here that the supposed nuclease activity of human and bacterial recombinant cyclophilins is due to contamination of the proteins by the host Escherichia coli endonuclease and is not an intrinsic property of these proteins.
Project description:Cyclophilins are ubiquitously expressed proteins that bind to prolines and can catalyse cis/trans isomerization of proline residues. There are 17 annotated members of the cyclophilin family in humans, ubiquitously expressed and localized variously to the cytoplasm, nucleus or mitochondria. Surprisingly, all eight of the nuclear localized cyclophilins are found associated with spliceosomal complexes. However, their particular functions within this context are unknown. We have therefore adapted three established assays for in vitro pre-mRNA splicing to probe the functional roles of nuclear cyclophilins in the context of the human spliceosome. We find that four of the eight spliceosom-associated cyclophilins exert strong effects on splicing in vitro. These effects are dose-dependent and, remarkably, uniquely characteristic of each cyclophilin. Using both qualitative and quantitative means, we show that at least half of the nuclear cyclophilins can act as regulatory factors of spliceosome function in vitro. The present work provides the first quantifiable evidence that nuclear cyclophilins are splicing factors and provides a novel approach for future work into small molecule-based modulation of pre-mRNA splicing.
Project description:Cyclophilins constitute a family of ubiquitous proteins that bind cyclosporin A (CsA), an immunosuppressant drug. Several of these proteins possess peptidyl-prolyl cis-trans isomerase (PPIase) activity that catalyzes the cis-trans isomerization of the peptide bond preceding a proline residue, essential for correct folding of the proteins. Compared to prokaryotes and other eukaryotes studied until now, the cyclophilin gene families in plants exhibit considerable expansion. With few exceptions, the role of the majority of these proteins in plants is still a matter of conjecture. However, recent studies suggest that cyclophilins are highly versatile proteins with multiple functionalities, and regulate a plethora of growth and development processes in plants, ranging from hormone signaling to the stress response. The present review discusses the implications of cyclophilins in different facets of cellular processes, particularly in the context of plants, and provides a glimpse into the molecular mechanisms by which these proteins fine-tune the diverse physiological pathways.
Project description:Bacterial extracellular nucleases have multiple functions in processes as diverse as nutrient acquisition, natural transformation, biofilm formation, or defense against neutrophil extracellular traps (NETs). Here we explored the properties of ExeM in Shewanella oneidensis MR-1, an extracellular nuclease, which is widely conserved among species of Shewanella, Vibrio, Aeromonas, and others. In S. oneidensis, ExeM is crucial for normal biofilm formation. In vitro activity measurements on heterologously produced ExeM revealed that this enzyme is a sugar-unspecific endonuclease, which requires Ca2+ and Mg2+/Mn2+ as co-factors for full activity. ExeM was almost exclusively localized to the cytoplasmic membrane fraction, even when a putative C-terminal membrane anchor was deleted. In contrast, ExeM was not detected in medium supernatants. Based on the results we hypothesize that ExeM predominantly interacts with DNA in close proximity to the cell, e.g., to promote biofilm formation and defense against NETs, or to control uptake of DNA.
Project description:Agrobacterium tumefaciens induces crown gall tumors on plants by transferring a nucleoprotein complex, the T-complex, from the bacterium to the plant cell. The T-complex consists of T-DNA, a single-stranded DNA segment of the tumor-inducing plasmid, VirD2, an endonuclease covalently bound to the 5' end of the T-DNA, and perhaps VirE2, a single-stranded DNA binding protein. The yeast two-hybrid system was used to screen for proteins interacting with VirD2 and VirE2 to identify components in Arabidopsis thaliana that interact with the T-complex. Three VirD2- and two VirE2-interacting proteins were identified. Here we characterize the interactions of VirD2 with two isoforms of Arabidopsis cyclophilins identified by using this analysis. The VirD2 domain interacting with the cyclophilins is distinct from the endonuclease, omega, and the nuclear localization signal domains. The VirD2-cyclophilin interaction is disrupted in vitro by cyclosporin A, which also inhibits Agrobacterium-mediated transformation of Arabidopsis and tobacco. These data strongly suggest that host cyclophilins play a role in T-DNA transfer.
Project description:Cyclophilins are prolyl isomerases with multitude of functions in different cellular processes and pathological conditions. Cyclophilin A (PpiA) of Mycobacterium tuberculosis is secreted during infection in intraphagosomal niche. However, our understanding about the evolutionary origin, secretory mechanism or the interactome of M. tuberculosis PpiA is limited. This study demonstrates through phylogenetic and structural analyses that PpiA has more proximity to human cyclophilins than the prokaryotic counterparts. We report a unique N-terminal sequence (MADCDSVTNSP) present in pathogenic mycobacterial PpiA and absent in non-pathogenic strains. This sequence stretch was shown to be essential for PpiA secretion. The overexpression of full-length PpiA from M. tuberculosis in non-pathogenic Mycobacterium smegmatis resulted in PpiA secretion while truncation of the N-terminal stretch obstructed the secretion. In addition, presence of an ESX pathway substrate motif in M. tuberculosis PpiA suggested possible involvement of Type VII secretion system. Site-directed mutagenesis of key residues in this motif in full-length PpiA also hindered the secretion in M. smegmatis. Bacterial two-hybrid screens with human lung cDNA library as target were utilized to identify interaction partners of PpiA from host repertoire, and a number of substrates with functional representation in iron storage, signal transduction and immune responses were detected. The extensive host interactome coupled with the sequence and structural similarity to human cyclophilins is strongly suggestive of PpiA being deployed by M. tuberculosis as an effector mimic against the host cyclophilins.
Project description:The role of Cdc45 in genomic duplication has remained unclear since its initial identification as an essential replication factor. Recent structural studies of Cdc45 and the evolutionarily-related archaeal GAN and bacterial RecJ nucleases have provided fresh insight into its function as co-activator of the MCM helicase. The CMG helicase of the last archaeal/eukaryotic ancestor might have harboured a single-stranded DNA nuclease activity, conserved in some modern archaea.
Project description:Protein folding within the endoplasmic reticulum is assisted by molecular chaperones and folding catalysts that include members of the protein-disulfide isomerase and peptidyl-prolyl isomerase families. In this report, we examined the contributions of the cyclophilin subset of peptidyl-prolyl isomerases to protein folding and identified cyclophilin C as an endoplasmic reticulum (ER) cyclophilin in addition to cyclophilin B. Using albumin and transferrin as models of cis-proline-containing proteins in human hepatoma cells, we found that combined knockdown of cyclophilins B and C delayed transferrin secretion but surprisingly resulted in more efficient oxidative folding and secretion of albumin. Examination of the oxidation status of ER protein-disulfide isomerase family members revealed a shift to a more oxidized state. This was accompanied by a >5-fold elevation in the ratio of oxidized to total glutathione. This "hyperoxidation" phenotype could be duplicated by incubating cells with the cyclophilin inhibitor cyclosporine A, a treatment that triggered efficient ER depletion of cyclophilins B and C by inducing their secretion to the medium. To identify the pathway responsible for ER hyperoxidation, we individually depleted several enzymes that are known or suspected to deliver oxidizing equivalents to the ER: Ero1αβ, VKOR, PRDX4, or QSOX1. Remarkably, none of these enzymes contributed to the elevated oxidized to total glutathione ratio induced by cyclosporine A treatment. These findings establish cyclophilin C as an ER cyclophilin, demonstrate the novel involvement of cyclophilins B and C in ER redox homeostasis, and suggest the existence of an additional ER oxidative pathway that is modulated by ER cyclophilins.
Project description:Cyclophilins are ubiquitous cis-trans-prolyl isomerases (PPIases) found in all kingdoms of life. Here, we identify a novel family of cyclophilins, termed AquaCyps, which specifically occurs in marine Alphaproteobacteria, but not in related terrestric species. In addition to a canonical PPIase domain, AquaCyps contain large extensions and insertions. The crystal structures of two representatives from Hirschia baltica, AquaCyp293 and AquaCyp300, reveal the formation of a compact domain, the NIC domain, by the N- and C-terminal extensions together with a central insertion. The NIC domain adopts a novel mixed alpha-helical, beta-sheet fold that is linked to the cyclophilin domain via a conserved disulfide bond. In its overall fold, AquaCyp293 resembles AquaCyp300, but the two proteins utilize distinct sets of active site residues, consistent with differences in their PPIase catalytic properties. While AquaCyp293 is a highly active general PPIase, AquaCyp300 is specific for hydrophobic substrate peptides and exhibits lower overall activity.
Project description:Despite an abundance of evidence supporting an important role for the cleavage of minor capsid protein L2 by cellular furin, direct cleavage of capsid-associated L2 during human papillomavirus 16 (HPV16) infection remains poorly characterized. The conserved cleavage site, close to the L2 N terminus, confounds observation and quantification of the small cleavage product by SDS-PAGE. To overcome this difficulty, we increased the size shift by fusing a compact protein domain, the Propionibacterium shermanii transcarboxylase domain (PSTCD), to the N terminus of L2. The infectious PSTCD-L2 virus displayed an appreciable L2 size shift during infection of HaCaT keratinocytes. Cleavage under standard cell culture conditions rarely exceeded 35% of total L2. Cleavage levels were enhanced by the addition of exogenous furin, and the absolute levels of infection correlated to the level of L2 cleavage. Cleavage occurred on both the HaCaT cell surface and extracellular matrix (ECM). Contrary to current models, experiments on the involvement of cyclophilins revealed little, if any, role for these cellular enzymes in the modulation of furin cleavage. HPV16 L2 contains two consensus cleavage sites, Arg5 (2RHKR5) and Arg12 (9RTKR12). Mutant PSTCD-L2 viruses demonstrated that although furin can cleave either site, cleavage must occur at Arg12, as cleavage at Arg5 alone is insufficient for successful infection. Mutation of the conserved cysteine residues revealed that the Cys22-Cys28 disulfide bridge is not required for cleavage. The PSTCD-L2 virus or similar N-terminal fusions will be valuable tools to study additional cellular and viral determinants of furin cleavage.Furin cleavage of minor capsid protein L2 during papillomavirus infection has been difficult to directly visualize and quantify, confounding efforts to study this important step of HPV infection. Fusion of a small protein domain to the N terminus greatly facilitates direct visualization of the cleavage product, revealing important characteristics of this critical process. Contrary to the current model, we found that cleavage is largely independent of cyclophilins, suggesting that cyclophilins act either in parallel to or downstream of furin to trigger exposure of a conserved N-terminal L2 epitope (RG-1) during infection. Based on this finding, we strongly caution against using L2 RG-1 epitope exposure as a convenient but indirect proxy of furin cleavage.
Project description:We report here a biochemical and structural characterization of domain 2 of the nonstructural 5A protein (NS5A) from the JFH1 Hepatitis C virus strain and its interactions with cyclophilins A and B (CypA and CypB). Gel filtration chromatography, circular dichroism spectroscopy, and finally NMR spectroscopy all indicate the natively unfolded nature of this NS5A-D2 domain. Because mutations in this domain have been linked to cyclosporin A resistance, we used NMR spectroscopy to investigate potential interactions between NS5A-D2 and cellular CypA and CypB. We observed a direct molecular interaction between NS5A-D2 and both cyclophilins. The interaction surface on the cyclophilins corresponds to their active site, whereas on NS5A-D2, it proved to be distributed over the many proline residues of the domain. NMR heteronuclear exchange spectroscopy yielded direct evidence that many proline residues in NS5A-D2 form a valid substrate for the enzymatic peptidyl-prolyl cis/trans isomerase (PPIase) activity of CypA and CypB.