The structural basis for the collagen processing by human P3H1/CRTAP/PPIB ternary complex.

Collagen posttranslational processing is crucial for its proper assembly and function. Disruption of collagen processing leads to tissue development and structure disorders like osteogenesis imperfecta (OI). OI-related collagen processing machinery includes prolyl 3-hydroxylase 1 (P3H1), peptidyl-prolyl cis-trans isomerase B (PPIB), and cartilage-associated protein (CRTAP), with their structural organization and mechanism unclear. We determine cryo-EM structures of the P3H1/CRTAP/PPIB complex. The active sites of P3H1 and PPIB form a face-to-face bifunctional reaction center, indicating a coupled modification mechanism. The structure of the P3H1/CRTAP/PPIB/collagen peptide complex reveals multiple binding sites, suggesting a substrate interacting zone. Unexpectedly, a dual-ternary complex is observed, and the balance between ternary and dual-ternary states can be altered by mutations in the P3H1/PPIB active site and the addition of PPIB inhibitors. These findings provide insights into the structural basis of collagen processing by P3H1/CRTAP/PPIB and the molecular pathology of collagen-related disorders.

Investigation of cross-opsonic effect leads to the discovery of PPIase-domain containing protein vaccine candidate to prevent infections by Gram-positive ESKAPE pathogens.

BACKGROUND: Enterococcus faecium and Staphylococcus aureus are the Gram-positive pathogens of the ESKAPE group, known to represent a great threat to human health due to their high virulence and multiple resistances to antibiotics. Combined, enterococci and S. aureus account for 26% of healthcare-associated infections and are the most common organisms responsible for blood stream infections. We previously showed that the peptidyl-prolyl cis/trans isomerase (PPIase) PpiC of E. faecium elicits the production of specific, opsonic, and protective antibodies that are effective against several strains of E. faecium and E. faecalis. Due to the ubiquitous characteristics of PPIases and their essential function within Gram-positive cells, we hypothesized a potential cross-reactive effect of anti-PpiC antibodies. RESULTS: Opsonophagocytic assays combined with bioinformatics led to the identification of the foldase protein PrsA as a new potential vaccine antigen in S. aureus. We show that PrsA is a stable dimeric protein able to elicit opsonic antibodies against the S. aureus strain MW2, as well as cross-binding and cross-opsonic in several S. aureus, E. faecium and E. faecalis strains. CONCLUSIONS: Given the multiple antibiotic resistances S. aureus and enterococci present, finding preventive strategies is essential to fight those two nosocomial pathogens. The study shows the potential of PrsA as an antigen to use in vaccine formulation against the two dangerous Gram-positive ESKAPE bacteria. Our findings support the idea that PPIases should be further investigated as vaccine targets in the frame of pan-vaccinomics strategy.

Positive charges promote the recognition of proteins by the chaperone SlyD from Escherichia coli.

SlyD is a widely-occurring prokaryotic FKBP-family prolyl isomerase with an additional chaperone domain. Often, such as in Escherichia coli, a third domain is found at its C-terminus that binds nickel and provides it for nickel-enzyme biogenesis. SlyD has been found to bind signal peptides of proteins that are translocated by the Tat pathway, a system for the transport of folded proteins across membranes. Using peptide arrays to analyze these signal peptide interactions, we found that SlyD interacted only with positively charged peptides, with a preference for arginines over lysines, and large hydrophobic residues enhanced binding. Especially a twin-arginine motif was recognized, a pair of highly conserved arginines adjacent to a stretch of hydrophobic residues. Using isothermal titration calorimetry (ITC) with purified SlyD and a signal peptide-containing model Tat substrate, we could show that the wild type twin-arginine signal peptide was bound with higher affinity than an RR>KK mutated variant, confirming that positive charges are recognized by SlyD, with a preference of arginines over lysines. The specific role of negative charges of the chaperone domain surface and of hydrophobic residues in the chaperone active site was further analyzed by ITC of mutated SlyD variants. Our data show that the supposed key hydrophobic residues of the active site are indeed crucial for binding, and that binding is influenced by negative charges on the chaperone domain. Recognition of positive charges is likely achieved by a large negatively charged surface region of the chaperone domain, which is highly conserved although individual positions are variable.

The chaperone PrsA2 regulates the secretion, stability, and folding of listeriolysin O during Listeria monocytogenes infection.

Pathogenic bacteria rely on secreted virulence factors to cause disease in susceptible hosts. However, in Gram-positive bacteria, the mechanisms underlying secreted protein activation and regulation post-membrane translocation remain largely unknown. Using proteomics, we identified several proteins that are dependent on the secreted chaperone PrsA2. We followed with phenotypic, biochemical, and biophysical assays and computational analyses to examine the regulation of a detected key secreted virulence factor, listeriolysin O (LLO), and its interaction with PrsA2 from the bacterial pathogen Listeria monocytogenes (Lm). Critical to Lm virulence is internalization by host cells and the subsequent action of the cholesterol-dependent pore-forming toxin, LLO, which enables bacterial escape from the host cell phagosome. Since Lm is a Gram-positive organism, the space between the cell membrane and wall is solvent exposed. Therefore, we hypothesized that the drop from neutral to acidic pH as the pathogen is internalized into a phagosome is critical to regulating the interaction of PrsA2 with LLO. Here, we demonstrate that PrsA2 directly interacts with LLO in a pH-dependent manner. We show that PrsA2 protects and sequesters LLO under neutral pH conditions where LLO can be observed to aggregate. In addition, we identify molecular features of PrsA2 that are required for interaction and ultimately the folding and activity of LLO. Moreover, protein-complex modeling suggests that PrsA2 interacts with LLO via its cholesterol-binding domain. These findings highlight a mechanism by which a Gram-positive secretion chaperone regulates the secretion, stability, and folding of a pore-forming toxin under conditions relevant to host cell infection. IMPORTANCE: Lm is a ubiquitous food-borne pathogen that can cause severe disease to vulnerable populations. During infection, Lm relies on a wide repertoire of secreted virulence factors including the LLO that enables the bacterium to invade the host and spread from cell to cell. After membrane translocation, secreted factors must become active in the challenging bacterial cell membrane-wall interface. However, the mechanisms required for secreted protein folding and function are largely unknown. Lm encodes a chaperone, PrsA2, that is critical for the activity of secreted factors. Here, we show that PrsA2 directly associates and protects the major Lm virulence factor, LLO, under conditions corresponding to the host cytosol, where LLO undergoes irreversible denaturation. Additionally, we identify molecular features of PrsA2 that enable its interaction with LLO. Together, our results suggest that Lm and perhaps other Gram-positive bacteria utilize secreted chaperones to regulate the activity of pore-forming toxins during infection.

Optimized expression of Peptidyl-prolyl cis/transisomerase cyclophilinB with prokaryotic toxicity from Sporothrix globosa.

Cyclophilin B (CypB), a significant member of immunophilins family with peptidyl-prolyl cis-trans isomerase (PPIase) activity, is crucial for the growth and metabolism of prokaryotes and eukaryotes. Sporothrix globosa (S. globosa), a principal pathogen in the Sporothrix complex, causes sporotrichosis. Transcriptomic analysis identified the cypB gene as highly expressed in S. globosa. Our previous study demonstrated that the recombinant Escherichia coli strain containing SgcypB gene failed to produce sufficient product when it was induced to express the protein, implying the potential toxicity of recombinant protein to the bacterial host. Bioinformatics analysis revealed that SgCypB contains transmembrane peptides within the 52 amino acid residues at the N-terminus and 21 amino acids near the C-terminus, and 18 amino acid residues within the cytoplasm. AlphaFold2 predicted a SgCypB 3D structure in which there is an independent PPIase domain consisting of a spherical extracellular part. Hence, we chose to express the extracellular domain to yield high-level recombinant protein with PPIase activity. Finally, we successfully produced high-yield, truncated recombinant CypB protein from S. globosa (SgtrCypB) that retained characteristic PPIase activity without host bacterium toxicity. This study presents an alternative expression strategy for proteins toxic to prokaryotes, such as SgCypB. ONE-SENTENCE SUMMARY: The recombinant cyclophilin B protein of Sporothrix globosa was expressed successfully by retaining extracellular domain with peptidyl-prolyl cis-trans isomerase activity to avoid toxicity to the host bacterium.

Bacterial Chaperone Domain Insertions Convert Human FKBP12 into an Excellent Protein-Folding Catalyst-A Structural and Functional Analysis.

Many folding enzymes use separate domains for the binding of substrate proteins and for the catalysis of slow folding reactions such as prolyl isomerization. FKBP12 is a small prolyl isomerase without a chaperone domain. Its folding activity is low, but it could be increased by inserting the chaperone domain from the homolog SlyD of E. coli near the prolyl isomerase active site. We inserted two other chaperone domains into human FKBP12: the chaperone domain of SlpA from E. coli, and the chaperone domain of SlyD from Thermococcus sp. Both stabilized FKBP12 and greatly increased its folding activity. The insertion of these chaperone domains had no influence on the FKBP12 and the chaperone domain structure, as revealed by two crystal structures of the chimeric proteins. The relative domain orientations differ in the two crystal structures, presumably representing snapshots of a more open and a more closed conformation. Together with crystal structures from SlyD-like proteins, they suggest a path for how substrate proteins might be transferred from the chaperone domain to the prolyl isomerase domain.

Nuclear Phosphoproteome Reveals Prolyl Isomerase PIN1 as a Modulator of Oncogene-Induced Senescence.

Mammalian cells possess intrinsic mechanisms to prevent tumorigenesis upon deleterious mutations, including oncogene-induced senescence (OIS). The molecular mechanisms underlying OIS are, however, complex and remain to be fully characterized. In this study, we analyzed the changes in the nuclear proteome and phosphoproteome of human lung fibroblast IMR90 cells during the progression of OIS induced by oncogenic RASG12V activation. We found that most of the differentially regulated phosphosites during OIS contained prolyl isomerase PIN1 target motifs, suggesting PIN1 is a key regulator of several promyelocytic leukemia nuclear body proteins, specifically regulating several proteins upon oncogenic Ras activation. We showed that PIN1 knockdown promotes cell proliferation, while diminishing the senescence phenotype and hallmarks of senescence, including p21, p16, and p53 with concomitant accumulation of the protein PML and the dysregulation of promyelocytic leukemia nuclear body formation. Collectively, our data demonstrate that PIN1 plays an important role as a tumor suppressor in response to oncogenic ER:RasG12V activation.

Molecular docking, 3D-QASR and molecular dynamics simulations of benzimidazole Pin1 inhibitors.

Pin1 (protein interacting with never-in-mitosis akinase-1) is a member of the family of peptidylprolyl cis-trans isomerases (PPIases) that specifically recognize and isomerize substrates containing phosphorylated Ser/Thr-Pro sequences. Pin1 is involved in many cellular processes and plays a key role in the cell cycle, transcriptional regulation, cell metabolism, proliferation and differentiation, and its abnormalities lead to degenerative and neoplastic diseases. Pin1 is highly expressed in human cancers and promotes the development of tumors by activating multiple oncogenes and inactivating multiple tumor suppressor genes, making it an attractive target for cancer therapy. In this study, we investigated the binding mechanism and conformational relationship between benzimidazole Pin1 inhibitors and Pin1 proteins by molecular docking, three-dimensional quantitative structure-activity relationship (3D-QSAR) modeling, binding free energy calculations and decomposition, and molecular dynamics simulations. Molecular docking and molecular dynamics simulations disclosed the most likely binding pose of benzimidazoles with the Pin1 protein. The results of 3D-QSAR modeling indicated that electrostatic fields, hydrophobic fields and hydrogen bonding play important roles in the binding process of inhibitors to proteins. The binding free energy calculations and energy decomposition indicated that Lys63, Arg69, Cys113, Leu122, Met130, and Ser154 may be key residues in the binding of benzimidazole-based inhibitors to the Pin1 protein. This study provides an important theoretical basis for the design and optimization of benzimidazole compounds.

Stabilization of Pin1 by USP34 promotes Ubc9 isomerization and protein sumoylation in glioma stem cells.

The peptidyl-prolyl cis-trans isomerase Pin1 is a pivotal therapeutic target in cancers, but the regulation of Pin1 protein stability is largely unknown. High Pin1 expression is associated with SUMO1-modified protein hypersumoylation in glioma stem cells (GSCs), but the underlying mechanisms remain elusive. Here we demonstrate that Pin1 is deubiquitinated and stabilized by USP34, which promotes isomerization of the sole SUMO E2 enzyme Ubc9, leading to SUMO1-modified hypersumoylation to support GSC maintenance. Pin1 interacts with USP34, a deubiquitinase with preferential expression and oncogenic function in GSCs. Such interaction is facilitated by Plk1-mediated phosphorylation of Pin1. Disruption of USP34 or inhibition of Plk1 promotes poly-ubiquitination and degradation of Pin1. Furthermore, Pin1 isomerizes Ubc9 to upregulate Ubc9 thioester formation with SUMO1, which requires CDK1-mediated phosphorylation of Ubc9. Combined inhibition of Pin1 and CDK1 with sulfopin and RO3306 most effectively suppresses orthotopic tumor growth. Our findings provide multiple molecular targets to induce Pin1 degradation and suppress hypersumoylation for cancer treatment.

Local Net Charge State of Collagen Triple Helix Is a Determinant of FKBP22 Binding to Collagen III.

Mutations in the FKBP14 gene encoding the endoplasmic reticulum resident collagen-related proline isomerase FK506 binding protein 22 kDa (FKBP22) result in kyphoscoliotic Ehlers-Danlos Syndrome (EDS), which is characterized by a broad phenotypic outcome. A plausible explanation for this outcome is that FKBP22 participates in the biosynthesis of subsets of collagen types: FKBP22 selectively binds to collagens III, IV, VI, and X, but not to collagens I, II, V, and XI. However, these binding mechanisms have never been explored, and they may underpin EDS subtype heterogeneity. Here, we used collagen Toolkit peptide libraries to investigate binding specificity. We observed that FKBP22 binding was distributed along the collagen helix. Further, it (1) was higher on collagen III than collagen II peptides and it (2) was correlated with a positive peptide charge. These findings begin to elucidate the mechanism by which FKBP22 interacts with collagen.

Characterization and regulation of salt upregulated cyclophilin from a halotolerant strain of Penicillium oxalicum.

Penicillium species are an industrially important group of fungi. Cyclophilins are ubiquitous proteins and several members of this family exhibit peptidyl-prolyl cis-trans isomerase (PPIase) activity. We had earlier demonstrated that the salt-induced PPIase activity in a halotolerant strain of P. oxalicum was associated with enhanced expression of a cyclophilin gene, PoxCYP18. Cloning and characterization of PoxCYP18 revealed that its cDNA consists of 522 bp encoding a protein of 173 amino acid residues, with predicted molecular mass and pI values of 18.91 kDa and 8.87, respectively. The recombinant PoxCYP18 can catalyze cis-trans isomerization of peptidyl-prolyl bond with a catalytic efficiency of 1.46 × 107 M-1 s-1 and is inhibited specifically only by cyclosporin A, with an inhibition constant of 5.04 ± 1.13 nM. PoxCYP18 consists of two cysteine residues at positions - 45 and - 170, and loses its activity under oxidizing conditions. Substitution of these residues alone or together by site-directed mutagenesis revealed that the PPIase activity of PoxCYP18 is regulated through a redox mechanism involving the formation of disulfide linkages. Heterologous expression of PoxCYP18 conferred enhanced tolerance to salt stress in transgenic E. coli cells, implying that this protein imparts protection to cellular processes against salt-induced damage.

The SlyD metallochaperone targets iron-sulfur biogenesis pathways and the TCA cycle.

IMPORTANCE: Correct folding of proteins represents a crucial step for their functions. Among the chaperones that control protein folding, the ubiquitous PPIases catalyze the cis/trans-isomerization of peptidyl-prolyl bonds. Only few protein targets of PPIases have been reported in bacteria. To fill this knowledge gap, we performed a large-scale two-hybrid screen to search for targets of the Escherichia coli and Helicobacter pylori SlyD PPIase-metallochaperone. SlyD from both organisms interacts with enzymes (i) containing metal cofactors, (ii) from the central metabolism tricarboxylic acid (TCA) cycle, and (iii) involved in the formation of the essential and ancestral Fe-S cluster cofactor. E. coli and H. pylori ∆slyD mutants present similar phenotypes of diminished susceptibility to antibiotics and to oxidative stress. In H. pylori, measurements of the intracellular ATP content, proton motive force, and activity of TCA cycle proteins suggest that SlyD regulates TCA cycle enzymes by controlling the formation of their indispensable Fe-S clusters.

Peptidyl-prolyl cis/trans isomerase Pin1 interacts with hepatitis B virus core particle, but not with HBc protein, to promote HBV replication.

Here, we demonstrate that the peptidyl-prolyl cis/trans isomerase Pin1 interacts noncovalently with the hepatitis B virus (HBV) core particle through phosphorylated serine/threonine-proline (pS/TP) motifs in the carboxyl-terminal domain (CTD) but not with particle-defective, dimer-positive mutants of HBc. This suggests that neither dimers nor monomers of HBc are Pin1-binding partners. The 162TP, 164SP, and 172SP motifs within the HBc CTD are important for the Pin1/core particle interaction. Although Pin1 dissociated from core particle upon heat treatment, it was detected as an opened-up core particle, demonstrating that Pin1 binds both to the outside and the inside of the core particle. Although the amino-terminal domain S/TP motifs of HBc are not involved in the interaction, 49SP contributes to core particle stability, and 128TP might be involved in core particle assembly, as shown by the decreased core particle level of S49A mutant through repeated freeze and thaw and low-level assembly of the T128A mutant, respectively. Overexpression of Pin1 increased core particle stability through their interactions, HBV DNA synthesis, and virion secretion without concomitant increases in HBV RNA levels, indicating that Pin1 may be involved in core particle assembly and maturation, thereby promoting the later stages of the HBV life cycle. By contrast, parvulin inhibitors and PIN1 knockdown reduced HBV replication. Since more Pin1 proteins bound to immature core particles than to mature core particles, the interaction appears to depend on the stage of virus replication. Taken together, the data suggest that physical association between Pin1 and phosphorylated core particles may induce structural alterations through isomerization by Pin1, induce dephosphorylation by unidentified host phosphatases, and promote completion of virus life cycle.

Oxygen-independent stabilization of HIF-2α in breast cancer through direct interaction with peptidyl-prolyl cis-trans isomerase NIMA-interacting 1.

Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1) isomerizes the nearby proline (Pro) residue when it detects phosphorylated serine (Ser) or threonine (Thr) of target proteins, altering their structure, stability, function, and interaction with other proteins. Hypoxia-inducible factor 2α (HIF-2α), a transcription factor that transactivates many oncogenic genes under hypoxic conditions, harbours the pSer/Thr-Pro motif. We found for the first time that Pin1 binds to HIF-2α physically in normoxic as well as hypoxic conditions in human breast cancer cells. The level of ubiquitinated HIF-2α was significantly raised by Pin1 knockdown, while expression of its mRNA transcript was unaffected. In agreement with this observation, the cycloheximide chase assay demonstrated that Pin1 prolonged the stability of HIF-2α. Serine 672, 696, and 790 of HIF-2α were found to undergo phosphorylation. Of these, the main amino acid involved in the Pin1 binding and HIF-2α stabilization was identified as serine 790, located in the nuclear export signal region of HIF-2α. The tissue array with human breast cancer specimens showed elevated expression of HIF-2α as well as Pin1 compared to adjacent normal tissues. Knockdown of Pin1 or HIF-2α diminished breast cancer cell migration and colony formation. In conclusion, Pin1 stabilizes HIF-2α through direct interaction, which contributes to the growth of breast cancer.

Association of peptidyl prolyl cis/trans isomerase Rrd1 with C terminal domain of RNA polymerase II.

The largest subunit of RNAPII extends as the conserved unstructured heptapeptide consensus repeats Y1S2P3T4S5P6S7 and their posttranslational modification, especially the phosphorylation state at Ser2, Ser5 and Ser7 of CTD recruits different transcription factors involved in transcription. In the current study, fluorescence anisotropy, pull down assay and molecular dynamics simulation studies employed to conclude that peptidyl-prolyl cis/trans-isomerase Rrd1 has strong affinity for unphosphorylated CTD rather than phosphorylated CTD for mRNA transcription. Rrd1 preferentially interacts with unphosphorylated GST-CTD in comparison to hyperphosphorylated GST-CTD in vitro. Fluorescence anisotropy revealed that recombinant Rrd1 prefers to bind unphosphorylated CTD peptide in comparison to phosphorylated CTD peptide. In computational studies, the RMSD of Rrd1-unphosphorylated CTD complex was greater than the RMSD of Rrd1-pCTD complex. During 50 ns MD simulation run Rrd1-pCTD complex get dissociated twice viz. 20 ns to 30 ns and 40 ns to 50 ns, while Rrd1-unpCTD complex remain stable throughout the process. Additionally, the Rrd1-unphosphorylated CTD complexes acquire comparatively higher number of H-bonds, water bridges and hydrophobic interactions occupancy than Rrd1-pCTD complex, concludes that the Rrd1 interacts more strongly with the unphosphorylated CTD than the pCTD.

Hepatic Pin1 Expression, Particularly in Nuclei, Is Increased in NASH Patients in Accordance with Evidence of the Role of Pin1 in Lipid Accumulation Shown in Hepatoma Cell Lines.

Our previous studies using rodent models have suggested an essential role for Pin1 in the pathogenesis of non-alcoholic steatohepatitis (NASH). In addition, interestingly, serum Pin1 elevation has been reported in NASH patients. However, no studies have as yet examined the Pin1 expression level in human NASH livers. To clarify this issue, we investigated the expression level and subcellular distribution of Pin1 in liver specimens obtained using needle-biopsy samples from patients with NASH and healthy liver donors. Immunostaining using anti-Pin1 antibody revealed the Pin1 expression level to be significantly higher, particularly in nuclei, in the livers of NASH patients than those of healthy donors. In the samples from patients with NASH, the amount of nuclear Pin1 was revealed to be negatively related to serum alanine aminotransferase (ALT), while tendencies to be associated with other serum parameters such as aspartate aminotransferase (AST) and platelet number were noted but did not reach statistical significance. Such unclear results and the lack of a significant relationship might well be attributable to our small number of NASH liver samples (n = 8). Moreover, in vitro, it was shown that addition of free fatty acids to medium induced lipid accumulation in human hepatoma HepG2 and Huh7 cells, accompanied with marked increases in nuclear Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1), in accordance with the aforementioned observations in human NASH livers. In contrast, suppression of Pin1 gene expression using siRNAs attenuated the free fatty acid-induced lipid accumulation in Huh7 cells. Taken together, these observations strongly suggest that increased expression of Pin1, particularly in hepatic nuclei, contributes to the pathogenesis of NASH with lipid accumulation.

The expression of prolyl isomerase Pin1 is expanded in the skin of patients with atopic dermatitis and facilitates IL-33 expression in HaCaT cells.

Atopic dermatitis (AD) is attributable to both a genetic predisposition and environmental factors. Among numerous cytokines involved in the pathogenesis of AD, interleukin-33 (IL-33), reportedly escaping exocytotically in response to a scratch, is abundantly expressed in the skin tissues of patients with AD and is postulated to induce inflammatory and autoimmune responses. In this study, we first demonstrated that peptidylprolyl cis/trans isomerase, NIMA-interacting 1 (Pin1), a unique enzyme which isomerizes the proline residues of target proteins, is abundantly expressed in keratinocytes, and that the areas where it is present in the skin tissues of AD patients became expanded due to hyperkeratosis. Thus, we investigated the effects of Pin1 on the regulation of IL-33 expression using the human keratinocyte cell line HaCaT. Interestingly, silencing of the Pin1 gene or treatment with Pin1 inhibitors dramatically reduced IL-33 expressions in HaCaT cells, although Pin1 overexpression did not elevate it. Subsequently, we showed that Pin1 binds to STAT1 and the nuclear factor-kappaB (NF-κB) subunit p65. Silencing the Pin1 gene with small interfering RNAs significantly reduced the phosphorylation of p65, while no marked effects of Pin1 on the STAT1 pathway were detected. Thus, it is likely that Pin1 contributes to increased expression of IL-33 via the NF-κB subunit p65 in HaCaT cells, at least modestly. Nevertheless, further study is necessary to demonstrate the pathogenic roles of Pin1 and IL-33 in AD development.

MicroRNA-150-5p inhibits the proliferation and invasion of human larynx epidermiod cancer cells though regulating peptidyl-prolyl cis/trans isomerase.

OBJECTIVE: This study aimed to investigate the molecular mechanism of miR-150-5p regulating the malignant biological behavior of Human Epidermoid cancer cell (HEp-2) by targeting peptidyl-prolyl cis/trans isomerase NIMA-Interacting-1 (PIN1). METHODS: Firstly, qRT-PCR and Western blot were adopted to detect the expression levels of miR-150-5p and PIN1 in cancer tissue and paracancerous tissues of patients with LSCC, and those in human bronchial epithelial cells (16 HBE) and HEp-2. Next, the targeted relationship between miR-150-5p and PIN1 was assessed by bioinformatics website and dual-luciferase reporter assay, followed by their correlation analysis. Besides, after interfering with miR-150-5p or PIN1 expression in HEp-2 cells, CCK-8, cell colony formation assay, and transwell assay were utilized to detect the proliferation, viability, and invasion of cells, respectively. Subsequently, the protein levels of MMP-2, MMP-9, and EMT-related proteins in HEp-2 cells were checked by Western blot. RESULTS: Expression of miR-150-5p was down-regulated in LSCC tissues and HEp-2 cells. Moreover, miR-150-5p suppressed proliferation and invasion of HEp-2 cells, affected protein expression related to MMP and EMT, thereby inhibiting development of cancer. The expression of PIN1 was significantly increased in cancer tissues and HEp-2 cells, and there was a targeted relationship and negative correlation between miR-150-5p and PIN1 in cancer tissue. However, overexpression of PIN1 could reverse the effect of miR-150-5p on the proliferation and invasion of HEp-2 cells. CONCLUSION: In a nutshell, there is a targeted relationship between PIN1 and miR-150-5p. Besides, miR-150-5p can inhibit the proliferation and invasion of HEp-2 cells by regulating the expression of PIN1.

Meddling with Metal Sensors: Fur-Family Proteins as Signaling Hubs.

The ferric uptake regulator (Fur) protein is the founding member of the FUR superfamily of metalloregulatory proteins that control metal homeostasis in bacteria. FUR proteins regulate metal homeostasis in response to the binding of iron (Fur), zinc (Zur), manganese (Mur), or nickel (Nur). FUR family proteins are generally dimers in solution, but the DNA-bound complex can involve a single dimer, a dimer-of-dimers, or an extended array of bound protein. Elevated FUR levels due to changes in cell physiology increase DNA occupancy and may also kinetically facilitate protein dissociation. Interactions between FUR proteins and other regulators are commonplace, often including cooperative and competitive DNA-binding interactions within the regulatory region. Further, there are many emerging examples of allosteric regulators that interact directly with FUR family proteins. Here, we focus on newly uncovered examples of allosteric regulation by diverse Fur antagonists (Escherichia coli YdiV/SlyD, Salmonella enterica EIIANtr, Vibrio parahaemolyticus FcrX, Acinetobacter baumannii BlsA, Bacillus subtilis YlaN, and Pseudomonas aeruginosa PacT) as well as one Zur antagonist (Mycobacterium bovis CmtR). Small molecules and metal complexes may also serve as regulatory ligands, with examples including heme binding to Bradyrhizobium japonicum Irr and 2-oxoglutarate binding to Anabaena FurA. How these protein-protein and protein-ligand interactions act in conjunction with regulatory metal ions to facilitate signal integration is an active area of investigation.

The metabolic crosstalk between PIN1 and the tumour microenvironment.

Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1) is a member of a family of peptidyl-prolyl isomerases that specifically recognizes and binds phosphoproteins, catalyzing the rapid cis-trans isomerization of phosphorylated serine/threonine-proline motifs, which leads to changes in the structures and activities of the targeted proteins. Through this complex mechanism, PIN1 regulates many hallmarks of cancer including cell autonomous metabolism and the crosstalk with the cellular microenvironment. Many studies showed that PIN1 is largely overexpressed in cancer turning on a set of oncogenes and abrogating the function of tumor suppressor genes. Among these targets, recent evidence demonstrated that PIN1 is involved in lipid and glucose metabolism and accordingly, in the Warburg effect, a characteristic of tumor cells. As an orchestra master, PIN1 finely tunes the signaling pathways allowing cancer cells to adapt and take advantage from a poorly organized tumor microenvironment. In this review, we highlight the trilogy among PIN1, the tumor microenvironment and the metabolic program rewiring.