A Redox-Sensitive Cysteine Is Required for PIN1At Function

Parvulins are ubiquitous peptidyl-prolyl isomerases (PPIases) required for protein folding and regulation. Among parvulin members, Arabidopsis PIN1At, human PIN1, and yeast ESS1 share a conserved cysteine residue but differ by the presence of an N-terminal WW domain, absent in PIN1At. In this study, we have explored whether the cysteine residue of Arabidopsis PIN1At is involved in catalysis and subject to oxidative modifications. From the functional complementation of yeast ess1 mutant, we concluded that the cysteine at position 69 is mandatory for PIN1At function in vivo, unless being replaced by an Asp which is found in a few parvulin members. This result correlates with a decrease of the in vitro PPIase activity of non-functional PIN1At cysteinic variants. A decrease of PIN1At activity was observed upon H2O2 treatment. The in vitro oxidation of cysteine 69, which has an acidic pKa value of 4.9, leads to the formation of covalent dimers that are reduced by thioredoxins, or to sulfinic or sulfonic acid forms at higher H2O2 excess. These investigations highlight the importance of the sole cysteine residue of PIN1At for activity. The reversible formation of an intermolecular disulfide bond might constitute a protective or regulatory mechanism under oxidizing conditions.

Genome-wide characterization of peptidyl-prolyl cis–trans isomerases in Penicillium and their regulation by salt stress in a halotolerant P. oxalicum

Peptidyl-prolyl cis–trans isomerases (PPIases) are the only class of enzymes capable of cis–trans isomerization of the prolyl peptide bond. The PPIases, comprising of different families viz., cyclophilins, FK506-binding proteins (FKBPs), parvulins and protein phosphatase 2A phosphatase activators (PTPAs), play essential roles in different cellular processes. Though PPIase gene families have been characterized in different organisms, information regarding these proteins is lacking in Penicillium species, which are commercially an important fungi group. In this study, we carried out genome-wide analysis of PPIases in different Penicillium spp. and investigated their regulation by salt stress in a halotolerant strain of Penicillium oxalicum. These analyses revealed that the number of genes encoding cyclophilins, FKBPs, parvulins and PTPAs in Penicillium spp. varies between 7–11, 2–5, 1–2, and 1–2, respectively. The halotolerant P. oxalicum depicted significant enhancement in the mycelial PPIase activity in the presence of 15% NaCl, thus, highlighting the role of these enzymes in salt stress adaptation. The stress-induced increase in PPIase activity at 4 and 10 DAI in P. oxalicum was associated with higher expression of PoxCYP18. Characterization of PPIases in Penicillium spp. will provide an important database for understanding their cellular functions and might facilitate their applications in industrial processes through biotechnological interventions.

Cyclophilin A inhibits RSV replication by binding to RSV-N through its PPIase activity

Human Respiratory syncytial virus (hRSV) is the most common pathogen which causes acute lower respiratory infection (ALRI) in infants. Recently, virus-host interaction is a hot spot of virus related research, and it needs to be further elaborated on its host interactions during RSV infection. Here, we found that RSV infection significantly increased the expression of cypA in clinical patients, mice or epithelial cells. Therefore, we evaluated the function of cypA in RSV replication and demonstrated that virus proliferation was accelerated in cypA knockdown host cells, but restrained in cypA overexpressed host cells. Furthermore, we proved that cypA limited RSV replication depending on its PPIase activity. Moreover, we performed liquid Chromatograph Mass Spectrometer and the results showed that cypA could interact with several viral proteins, such as RSV-N, RSV-P and RSV-M2-1. Finally, the interaction between cypA and RSV-N was certificated by co-immunoprecipitation and immunofluorescence. Those results provided strong evidence that cypA may play an inhibitory role in RSV replication through interacting with RSV-N via its PPIase activity. IMPORTANCE RSV-N, packed the viral genome to form the ribonuceloprotein complex (RNP), which is recognized by RSV RNA dependent RNA polymerase (RdRp) complex to initiate the viral replication and transcription, plays an indispensable role in viral biosynthesis process. CypA, binding to RSV-N, may impair this function by weakening the interaction between RSV-N and RSV-P, thus leading to decrease viral production. Our research provides a novel insight into cypA antiviral function by binding to viral capsid protein to inhibit viral replication, which may be helpful for new antiviral drug exploration.

Phenotypic characters and identification CYPs (Cyclophilin) gene in Cucumis melo L. cv. Gama Melon Parfum

Maryanto SD, Wibowo WA, Daryono BS. 2021. Phenotypic characters and identification CYPs (Cyclophilin) gene in Cucumis melo L. cv. Gama Melon Parfum. Biodiversitas 22: 3007-3014. Cucumis melo L. cv. Gama Melon Parfum is a new cultivar with a very strong fragrance as its main character. As a new cultivar with unique characters, it is necessary to characterize phenotype and molecular related to the fragrant aroma. The research aimed to study the phenotypic of the fruit of C. melo cv. Gama Melon Parfum (F3) and to identify the CYPs gene as one of the genes that act in encoding volatile compounds. Analysis of qualitative characters was based on International Plant Genetic Resources Institute (IPGRI) and Plant Variety Protection (PVP), phenotypic characters in melons observed by abiding the Rules for Registration of Varieties from the Indonesian Minister of Agriculture Decree No. 700/Kpts/OT.320/D/12/2011, while analysis of quantitative characters was using the ANOVA methods and software PKBT-STAT-2. The methods of molecular characterization included RNA isolation, cDNA synthesis used Reverse Transcriptase-PCR, amplification of DNA target used PCR, visualization of DNA target used electrophoresis, and DNA sequencing. Analysis of the in silico approach was carried out on the CuGenDB Melon database. Analysis of protein sequences and classification was obtained from InterPro. Phylogeny analysis using MEGA-X Software. The results were 18 qualitative characters and 11 quantitative characters were stable and uniform, whereas the molecular characterization of the genes was predicted Cyclophilin with peptidylprolyl isomerase (PPIase) activity and located in chromosome 1 (17059021-17058899).

FKBP52 overexpression accelerates hippocampal-dependent memory impairments in a tau transgenic mouse model

Abnormal accumulation of hyperphosphorylated tau induces pathogenesis in neurodegenerative diseases, like Alzheimer’s disease. Molecular chaperones with peptidyl-prolyl cis/trans isomerase (PPIase) activity are known to regulate these processes. Previously, in vitro studies have shown that the 52 kDa FK506-binding protein (FKBP52) interacts with tau inducing its oligomerization and fibril formation to promote toxicity. Thus, we hypothesized that increased expression of FKBP52 in the brains of tau transgenic mice would alter tau phosphorylation and neurofibrillary tangle formation ultimately leading to memory impairments. To test this, tau transgenic (rTg4510) and wild-type mice received bilateral hippocampal injections of virus overexpressing FKBP52 or GFP control. We examined hippocampal-dependent memory, synaptic plasticity, tau phosphorylation status, and neuronal health. This work revealed that rTg4510 mice overexpressing FKBP52 had impaired spatial learning, accompanied by long-term potentiation deficits and hippocampal neuronal loss, which was associated with a modest increase in total caspase 12. Together with previous studies, our findings suggest that FKBP52 may sensitize neurons to tau-mediated dysfunction via activation of a caspase-dependent pathway, contributing to memory and learning impairments.

Genome-Wide Characterization of Peptidyl-Prolyl Cis-Trans Isomerases in Penicillium and Their Regulation By Salt Stress in a Halotolerant P. Oxalicum

Peptidyl-prolyl cis-trans isomerases (PPIases) are the only class of enzymes capable of cis-trans isomerization of the prolyl peptide bond. The PPIases, comprising of different families viz., cyclophilins, FK506-binding proteins (FKBPs), parvulins and protein phosphatase 2A phosphatase activators (PTPAs), play essential roles in different cellular processes. Though PPIase gene families have been characterized in different organisms, information regarding these proteins lacks in Penicillium species, which are commercially an important fungi group. In this study, we carried out genome-wide analysis of PPIases in different Penicillium spp. and investigated their regulation by salt stress in a halotolerant strain of Penicillium oxalicum. These analyses revealed that the number of genes encoding cyclophilins, FKBPs, parvulins and PTPAs in Penicillium spp. varies between 7-11, 2-5, 1-2, and 1-2, respectively. The halotolerant P. oxalicum depicted significant enhancement in the mycelial PPIase activity in the presence of 15% NaCl, thus, highlighting the role of these enzymes in salt stress adaptation. The PPIase activity in P. oxalicum was associated with the expression of PoxCYP18, PoxCYP23, PoxCYP41, PoxFKBP12-2, and PoxFKBP52 genes. Characterization of PPIases in Penicillium spp. will provide an important database for understanding their cellular functions and might facilitate their applications in industrial processes through biotechnological interventions.

Structure of an Hsp90-immunophilin complex reveals cochaperone recognition of the client-maturation state

SummaryThe Hsp90 chaperone promotes the folding and activation of hundreds of client proteins in the cell through an ATP-dependent conformational cycle guided by distinct cochaperone regulators. The FKBP51 immunophilin binds Hsp90 with its tetratricopeptide repeat (TPR) domain and catalyzes peptidyl-prolyl isomerase (PPIase) activity during the folding of kinases, nuclear receptors and tau. Here we have determined the cryo-EM structure of the human Hsp90:FKBP51:p23 complex to 3.3 Å that, together with mutagenesis and crosslinking analysis, reveals the basis for cochaperone binding to Hsp90 during client maturation. A helix extension in the TPR functions as a key recognition element, interacting across the Hsp90 C-terminal dimer interface presented in the closed, ATP conformation. The PPIase domain is positioned along the middle domain, adjacent Hsp90 client binding sites, while a single p23 makes stabilizing interactions with the N-terminal dimer. With this architecture, FKBP51 could thereby act on specific client residues presented during Hsp90-catalyzed remodeling.

A novel mode of control of nickel uptake by a multifunctional metallochaperone

Cellular metal homeostasis is a critical process for all organisms, requiring tight regulation. In the major pathogen Helicobacter pylori, the acquisition of nickel is an essential virulence determinant as this metal is a cofactor for the acid-resistance enzyme, urease. Nickel uptake relies on the NixA permease and the NiuBDE ABC transporter. Till now, bacterial metal transporters were reported to be controlled at their transcriptional level. Here we uncovered post-translational regulation of the essential Niu transporter in H. pylori. Indeed, we demonstrate that SlyD, a protein combining peptidyl-prolyl isomerase (PPIase), chaperone, and metal-binding properties, is required for the activity of the Niu transporter. Using two-hybrid assays, we found that SlyD directly interacts with the NiuD permease subunit and identified a motif critical for this contact. Mutants of the different SlyD functional domains were constructed and used to perform in vitro PPIase activity assays and four different in vivo tests measuring nickel intracellular accumulation or transport in H. pylori. In vitro, SlyD PPIase activity is down-regulated by nickel, independently of its C-terminal region reported to bind metals. In vivo, a role of SlyD PPIase function was only revealed upon exposure to high nickel concentrations. Most importantly, the IF chaperone domain of SlyD was shown to be mandatory for Niu activation under all in vivo conditions. These data suggest that SlyD is required for the active functional conformation of the Niu permease and regulates its activity through a novel mechanism implying direct protein interaction, thereby acting as a gatekeeper of nickel uptake. Finally, in agreement with a central role of SlyD, this protein is essential for the colonization of the mouse model by H. pylori.

Multicopy Suppressor Analysis of Strains Lacking Cytoplasmic Peptidyl-Prolyl cis/trans Isomerases Identifies Three New PPIase Activities in Escherichia coli That Includes the DksA Transcription Factor

Consistent with a role in catalyzing rate-limiting step of protein folding, removal of genes encoding cytoplasmic protein folding catalysts belonging to the family of peptidyl-prolyl cis/trans isomerases (PPIs) in Escherichia coli confers conditional lethality. To address the molecular basis of the essentiality of PPIs, a multicopy suppressor approach revealed that overexpression of genes encoding chaperones (DnaK/J and GroL/S), transcriptional factors (DksA and SrrA), replication proteins Hda/DiaA, asparatokinase MetL, Cmk and acid resistance regulator (AriR) overcome some defects of Δ6ppi strains. Interestingly, viability of Δ6ppi bacteria requires the presence of transcriptional factors DksA, SrrA, Cmk or Hda. DksA, MetL and Cmk are for the first time shown to exhibit PPIase activity in chymotrypsin-coupled and RNase T1 refolding assays and their overexpression also restores growth of a Δ(dnaK/J/tig) strain, revealing their mechanism of suppression. Mutagenesis of DksA identified that D74, F82 and L84 amino acid residues are critical for its PPIase activity and their replacement abrogated multicopy suppression ability. Mutational studies revealed that DksA-mediated suppression of either Δ6ppi or ΔdnaK/J is abolished if GroL/S and RpoE are limiting, or in the absence of either major porin regulatory sensory kinase EnvZ or RNase H, transporter TatC or LepA GTPase or Pi-signaling regulator PhoU.

Targeting of parvulin interactors by diazirine mediated cross-linking discloses a cellular role of human Par14/17 in actin polymerization

The peptidyl-prolyl cis/trans isomerases (PPIases) Parvulin 14 (Par14) and Parvulin 17 (Par17) result from alternative transcription initiation of the PIN4 gene. Whereas Par14 is present in all metazoan, Par17 is only expressed in Hominidae. Par14 resides mainly within the cellular nucleus, while Par17 is translocated into mitochondria. Using photo-affinity labeling, cross-linking and mass spectrometry (MS) we identified binding partners for both enzymes from HeLa lysates and disentangled their cellular roles. Par14 is involved in biogenesis of ribonucleoprotein (RNP)-complexes, RNA processing and DNA repair. Its elongated isoform Par17 participates in protein transport/translocation and in cytoskeleton organization. Nuclear magnetic resonance (NMR) spectroscopy reveals that Par17 binds to β-actin with its N-terminal region, while both parvulins initiate actin polymerization depending on their PPIase activity as monitored by fluorescence spectroscopy. The knockdown (KD) of Par17 in HCT116 cells results in a defect in cell motility and migration.