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.

Cyclophilins and Their Roles in Hepatitis C Virus and Flavivirus Infections: Perspectives for Novel Antiviral Approaches

Cyclophilins are cellular peptidyl-prolyl isomerases that play an important role in viral infections, with demonstrated roles in the replication of hepatitis C virus (HCV) and other viruses in the Flaviviridae family, such as dengue virus (DENV) and yellow fever virus (YFV). Here, we discuss the roles of cyclophilins in HCV infection and provide a comprehensive overview of the mechanisms underlying the requirement for cyclophilins during HCV replication. Notably, cyclophilin inhibitor therapy has been demonstrated to be effective in reducing HCV replication in chronically infected patients. While the roles of cyclophilins are relatively well-understood for HCV infection, cyclophilins are more recently emerging as host factors for flavivirus infection as well, providing potential new therapeutic avenues for these viral infections which currently lack antiviral therapies. However, further studies are required to elucidate the roles of cyclophilins in flavivirus replication. Here, we review the current knowledge of the role of cyclophilins in HCV infection to provide a conceptual framework to understand how cyclophilins may contribute to other viral infections, such as DENV and YFV. Improved understanding of the roles of cyclophilins in viral infection may open perspectives for the development of cyclophilin inhibitors as effective antiviral therapeutics for HCV and related viruses.

BING, a novel antimicrobial peptide isolated from Japanese medaka plasma, targets bacterial envelope stress response by suppressing cpxR expression

Antimicrobial peptides (AMPs) have emerged as a promising alternative to small molecule antibiotics. Although AMPs have previously been isolated in many organisms, efforts on the systematic identification of AMPs in fish have been lagging. Here, we collected peptides from the plasma of medaka (Oryzias latipes) fish. By using mass spectrometry, 6399 unique sequences were identified from the isolated peptides, among which 430 peptides were bioinformatically predicted to be potential AMPs. One of them, a thermostable 13-residue peptide named BING, shows a broad-spectrum toxicity against pathogenic bacteria including drug-resistant strains, at concentrations that presented relatively low toxicity to mammalian cell lines and medaka. Proteomic analysis indicated that BING treatment induced a deregulation of periplasmic peptidyl-prolyl isomerases in gram-negative bacteria. We observed that BING reduced the RNA level of cpxR, an upstream regulator of envelope stress responses. cpxR is known to play a crucial role in the development of antimicrobial resistance, including the regulation of genes involved in drug efflux. BING downregulated the expression of efflux pump components mexB, mexY and oprM in P. aeruginosa and significantly synergised the toxicity of antibiotics towards these bacteria. In addition, exposure to sublethal doses of BING delayed the development of antibiotic resistance. To our knowledge, BING is the first AMP shown to suppress cpxR expression in Gram-negative bacteria. This discovery highlights the cpxR pathway as a potential antimicrobial target.

Identification of Salmonella Typhimurium Peptidyl-prolyl cis-trans Isomerase B (PPIase B) and Assessment of their Role in the Protein Folding

Background: Peptidyl-prolyl cis-trans isomerase (PPIases) enzyme plays a vital role in protein folding. It catalyses the cis-trans isomerisation of peptide bonds, an essential step for newly synthesized protein to acquire its correct functional conformation in both prokaryotes and eukaryotes. Objective: The present study showed the biochemical and molecular characterisation of cyclophilins (PpiB), a type of peptidyl-prolyl isomerases proteins from the pathogenic bacteria Salmonella Typhimurium. Methods: Salmonella Typhimurium is one of the leading serovars responsible for human and animal salmonellosis globally, with the majority of human cases originating through the food chain. Here successful expression and purification of PpiB protein have been demonstrated and LC-MS based analyses showed high protein score and similarity with other PPi protein. Further the enzymatic activity of the purified recombinant PpiB was determined using Succinyl-Ala-Phe-Pro- Phe-p nitroanilide as substrate and enzyme-catalysed reaction. Result: Km and Vmax were calculated and found to be Vm = 1.023 ± .06400 min/μg, Km = 0.6219 ± 0.1701 μM, respectively. We have reported for the first time the presence of Salmonella PPIase-B (PpiB) protein isoforms in salmonella genome having PPi activity. Conclusion: Taken together, our data clearly showed that Salmonella Cyclophilin B (PpiB) protein is active and involved in diverse biological processes and highly similar to the different domain of Cyclophilin proteins.

Molecular Chaperones in Cancer Stem Cells: Determinants of Stemness and Potential Targets for Antitumor Therapy

Cancer stem cells (CSCs) are a great challenge in the fight against cancer because these self-renewing tumorigenic cell fractions are thought to be responsible for metastasis dissemination and cases of tumor recurrence. In comparison with non-stem cancer cells, CSCs are known to be more resistant to chemotherapy, radiotherapy, and immunotherapy. Elucidation of mechanisms and factors that promote the emergence and existence of CSCs and their high resistance to cytotoxic treatments would help to develop effective CSC-targeting therapeutics. The present review is dedicated to the implication of molecular chaperones (protein regulators of polypeptide chain folding) in both the formation/maintenance of the CSC phenotype and cytoprotective machinery allowing CSCs to survive after drug or radiation exposure and evade immune attack. The major cellular chaperones, namely heat shock proteins (HSP90, HSP70, HSP40, HSP27), glucose-regulated proteins (GRP94, GRP78, GRP75), tumor necrosis factor receptor-associated protein 1 (TRAP1), peptidyl-prolyl isomerases, protein disulfide isomerases, calreticulin, and also a transcription heat shock factor 1 (HSF1) initiating HSP gene expression are here considered as determinants of the cancer cell stemness and potential targets for a therapeutic attack on CSCs. Various approaches and agents are discussed that may be used for inhibiting the chaperone-dependent development/manifestations of cancer cell stemness.

The peptidyl-prolyl isomerases FKBP15-1 and FKBP15-2 negatively regulate lateral root development by repressing a vacuolar invertase in Arabidopsis

Background Lateral root (LR) architecture determines the efficiency of nutrient absorption and anchors the plant. Internal auxin regulatory mechanisms that control the development of LR have been identified, but how external nutrients influence lateral root development remains elusive. Results We have characterized the functions of the FK506-binding proteins FKBP15-1 and FKBP15-2 in Arabidopsis. FKBP genes are mainly expressed in the vascular bundle of the root basal meristem region, and the FKBP proteins are localized to the endoplasmic reticulum. Co-IP and BIFC assays showed that FKBP15-1 and FKBP15-2 interact with the vacuolar invertase 2 (VIN2). Compared to Col-0 and the single mutants, the double mutant fkbp15-1fkbp15-2 had more LRs and LR initiation density, and possessed higher sucrose catalytic activity. Moreover, VIN2 can complement the phenotype of increased LRs in the fkbp15-1fkbp15-2 double mutant. Conclusion Our results indicate that FKBP15-1 and FKBP15-2 together participate in the control of LR numbers by regulating the enzyme activity of VIN2. Due to the activity of peptidylprolyl cis-trans isomerases owned by FKBP family proteins, our results provide a clue to further analysis the interplay between lateral root development and protein modification.

Quantification of reaction cycle parameters for an essential molecular switch in an auxin-responsive transcription circuit in rice

Protein-based molecular switches play critical roles in biological processes. The importance of the prolyl cis−trans switch is underscored by the ubiquitous presence of peptidyl prolyl isomerases such as cyclophilins that accelerate the intrinsically slow isomerization rate. In rice, a tryptophan−proline (W-P) cis−trans switch in transcription repressor protein OsIAA11 along with its associated cyclophilin LRT2 are essential components in a negative feedback gene regulation circuit that controls lateral root initiation in response to the plant hormone auxin. Importantly, no quantitative characterizations of the individual (microscopic) thermodynamic and kinetic parameters for any cyclophilin-catalyzed W-P isomerization have been reported. Here we present NMR studies that determine and independently validate these parameters for LRT2 catalysis of the W-P motif in OsIAA11, providing predictive power for understanding the role of this switch in the auxin-responsive circuit and the resulting lateral rootless phenotype in rice. We show that the observed isomerization rate is linearly dependent on LRT2 concentration but is independent of OsIAA11 concentration over a wide range, and LRT2 is optimally tuned to maintain OsIAA11 at its cis−trans equilibrium to supply the slower downstream cis-specific proteasomal degradation with maximal OsIAA11 substrate. This indicates that accelerating the LRT2-catalyzed isomerization would not accelerate OsIAA degradation, whereas decreasing this rate via targeted mutation could reveal relationships between circuit dynamics and lateral root development. Moreover, we show that sequences flanking the highly conserved Aux/IAA W-P motif do not impact LRT2 catalysis, suggesting that the parameters determined here are broadly applicable across highly conserved cyclophilins and their Aux/IAA targets.

Structural and Functional Insights into Human Nuclear Cyclophilins

The peptidyl prolyl isomerases (PPI) of the cyclophilin type are distributed throughout human cells, including eight found solely in the nucleus. Nuclear cyclophilins are involved in complexes that regulate chromatin modification, transcription, and pre-mRNA splicing. This review collects what is known about the eight human nuclear cyclophilins: peptidyl prolyl isomerase H (PPIH), peptidyl prolyl isomerase E (PPIE), peptidyl prolyl isomerase-like 1 (PPIL1), peptidyl prolyl isomerase-like 2 (PPIL2), peptidyl prolyl isomerase-like 3 (PPIL3), peptidyl prolyl isomerase G (PPIG), spliceosome-associated protein CWC27 homolog (CWC27), and peptidyl prolyl isomerase domain and WD repeat-containing protein 1 (PPWD1). Each “spliceophilin” is evaluated in relation to the spliceosomal complex in which it has been studied, and current work studying the biological roles of these cyclophilins in the nucleus are discussed. The eight human splicing complexes available in the Protein Data Bank (PDB) are analyzed from the viewpoint of the human spliceophilins. Future directions in structural and cellular biology, and the importance of developing spliceophilin-specific inhibitors, are considered.