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Peptidyl prolyl isomerases (PPIases) accelerate the rate limiting step of protein folding by catalyzing cis/trans isomerization of peptidyl prolyl bonds. The larger PPIases have been shown to be multi-domain proteins, with functions other than isomerization of the proline-containing peptide bond. Recently, a few smaller PPIases have also been described for their ability to stabilize folding intermediates. The yeast Fpr1 (FK506-sensitive proline rotamase) is a homologue of the mammalian prolyl isomerase FKBP12 (FK506-binding protein of 12 kDa). Its ability to stabilize stressed cellular proteins has not been reported yet. We had earlier reported upregulation of Fpr1 in yeast cells exposed to proteotoxic stress conditions. In this work, we show that yeast Fpr1 exhibits characteristics typical of a general chaperone of the proteostasis network. Aggregation of mutant huntingtin fragment was higher in Fpr1 -deleted as compared to parental yeast cells. Overexpression of Fpr1 led to reduced protein aggregation by decreasing the amount of oligomers and diverting the aggregation pathway towards the formation of detergent-soluble species. This correlated well with higher survival of these cells. Purified and enzymatically active yeast Fpr1 was able to inhibit aggregation of mutant huntingtin fragment and luciferase in vitro in a concentration-dependent manner; suggesting a direct action for aggregation inhibitory action of Fpr1. Overexpression of yeast Fpr1 was able to protect E. coli cells against thermal shock. This work establishes the role of Fpr1 in the protein folding network and will be used for the identification of novel pharmacological leads in disease conditions. [ABSTRACT FROM AUTHOR]

Objective: The aim of the present study was to construct a prognostic model based on the peptidyl prolyl cis – trans isomerase gene signature and explore the prognostic value of this model in patients with hepatocellular carcinoma. Methods: The transcriptome and clinical data of hepatocellular carcinoma patients were downloaded from The Cancer Genome Atlas and the International Cancer Genome Consortium database as the training set and validation set, respectively. Peptidyl prolyl cis – trans isomerase gene sets were obtained from the Molecular Signatures Database. The differential expression of peptidyl prolyl cis – trans isomerase genes was analyzed by R software. A prognostic model based on the peptidyl prolyl cis – trans isomerase signature was established by Cox, Lasso, and stepwise regression methods. Kaplan–Meier survival analysis was used to evaluate the prognostic value of the model and validate it with an independent external data. Finally, nomogram and calibration curves were developed in combination with clinical staging and risk score. Results: Differential gene expression analysis of hepatocellular carcinoma and adjacent tissues showed that there were 16 upregulated genes. A prognostic model of hepatocellular carcinoma was constructed based on three gene signatures by Cox, Lasso, and stepwise regression analysis. The Kaplan–Meier curve showed that hepatocellular carcinoma patients in high-risk score group had a worse prognosis (p < 0.05). The receiver operating characteristic curve revealed that the area under curve values of predicting the survival rate at 1, 2, 3, 4, and 5 years were 0.725, 0.680, 0.644, 0.630, and 0.639, respectively. In addition, the evaluation results of the model by the validation set were basically consistent with those of the training set. A nomogram incorporating clinical stage and risk score was established, and the calibration curve matched well with the diagonal. Conclusion: A prognostic model based on 3 peptidyl prolyl cis – trans isomerase gene signatures is expected to provide reference for prognostic risk stratification in patients with hepatocellular carcinoma. [ABSTRACT FROM AUTHOR]

Defects of autophagy-lysosomal protein degradation are thought to contribute to the pathogenesis of several neurodegenerative diseases, and the accumulation of aggregation prone proteins such as MAPT/Tau in Alzheimer disease (AD). We previously showed the localization of the immunophilin FKBP4/FKBP52 in the lysosomal system of healthy human neurons suggesting its possible role in lysosome function. We also showed that decreased FKBP4 levels in AD brain neurons correlate with abnormal MAPT accumulation and aggregation. In this study, we demonstrate that FKBP4 decrease in a human neuronal cell line (SH-SY5Y) and in dorsal root ganglion (DRG) neurons from human MAPTP301S transgenic mice affected the function of the autophagy-lysosomal system under MAPT induced proteotoxic stress conditions. We show that acute MAPT accumulation in SH-SY5Y cells induced perinuclear clustering of lysosomes, triggered FKBP4 localization around the clusters and its colocalization with MAPT and MAP1LC3/LC3-positive autophagic vesicles; a similar FKBP4 localization was detected in some AD brain neurons. We demonstrate that FKBP4 decrease altered lysosomal clustering along with MAPT and MAP1LC3 secretion increase. Although ectopic FKBP4 expression could not induce autophagy under our experimental conditions, it prevented MAPT secretion after MAPT accumulation in SH-SY5Y cells implying a regulatory role of FKBP4 on MAPT secretion. Finally, we observe that FKBP4 deficiency decreased MAP1LC3-II expression and provoked MAPT accumulation during long-term stress in mouse DRG neurons. We hypothesize that the abnormal FKBP4 decrease observed in AD brain neurons might hinder autophagy efficiency and contribute to the progression of the tauopathy by modulating MAPT secretion and accumulation during MAPT pathogenesis. Abbreviations: AD: Alzheimer disease; AKT/protein kinase B: AKT serine/threonine kinase; ALP: Autophagy-lysosomal pathway; ATG: autophagy-related; BafA1: bafilomycin A1; CQ: chloroquine; CTSD: cathepsin D; DIV: days in vitro; DRG: dorsal root ganglion neurons; Dox: doxycycline; DNAJC5: DnaJ heat shock protein family (Hsp40) member C5; EL: empty lentiviral vectors; ENO2/NSE: enolase 2, gamma neuronal; FKBP4/FKBP52: FKBP prolyl isomerase 4; FTLD-Tau: frontotemporal lobar degeneration with Tau pathology; GFP: green fluorescent protein; LAMP1: lysosomal associated membrane protein 1; LDH: lactate dehydrogenase; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAPT/Tau: microtubule associated protein tau; MTT: tetrazolium salt; NFTs: neurofibrillary tangles; RPE-1: retinal pigment epithelial cells; shRNA: small-hairpin ribonucleic acid; SQSTM1/p62: sequestosome 1; SD: standard deviation; SEM: standard error of the mean; SH-SY5Y: human neuroblastoma cells; Sh1 or Sh2: Lentiviral shRNA vectors inducing FKBP4 decrease; SH-52GFP: MAPT/Tau-inducible SH-SY5Y cell line constitutively expressing FKBP4-GFP; TUBB3/βIII tubulin: tubulin beta 3 class III; UPS: ubiquitin-proteasome system [ABSTRACT FROM AUTHOR]

[Display omitted] • Knockdown of Ppic attenuates CCl 4 -caused fibrotic alterations within livers and hepatic damage. • Ppic knockdown attenuates TGF-β-induced mouse hepatic stellate cell activation. • miR-137-3p directly targets and negatively regulates Ppic expression. Hepatic stellate cell activation, characterized by hyperproliferation and increased release of collagens, is a critical event during the initiation and development of hepatic fibrosis. The deregulated genes among different expression profiles based on online datasets were analyzed, attempting to identify novel potential biomarkers and treatment targets for hepatic fibrosis. The abnormal upregulation of mouse peptidylprolyl isomerase C (Ppic) within the CCl 4 -caused hepatic fibrosis model in mice was identified according to bioinformatics and experimental analyses. The knockdown of Ppic in the CCl 4 -caused liver fibrosis murine model significantly improved CCl 4 -caused liver damage, decreased the fibrotic area, reduced ECM deposition, and reduced the hydroxyproline levels. The knockdown of Ppic in TGF-β-stimulated mouse hepatic stellate cells inhibited cell proliferation and decreased ECM levels. Through direct targeting, miR-137-3p negatively regulated Ppic expression. Contrastingly to Ppic knockdown, miR-137-3p inhibition further promoted cell proliferation and boosted ECM levels; the effects of miR-137-3p inhibition could be partially reversed by Ppic knockdown. Altogether, mmu-miR-137-3p directly targets Ppic and forms a regulatory axis with Ppic , modulating CCl 4 -caused hepatic fibrosis in mice and TGF-β-caused mouse hepatic stellate cell activation. [ABSTRACT FROM AUTHOR]

Diabetic nephropathy is a complication of diabetes mellitus (both T1DM and T2DM), caused by microvascular changes and can lead to kidney failure and cardiovascular disease. The aim of this study is to evaluate the diagnostic value of cyclophilin A as a biomarker for the detection of diabetic nephropathy. The sample consisted of 119 T2DM patients, which included 65 females and 54 males whose ages ranged from 33 to 73.The participants were divided into three groups based on their urinary albumin to creatinine ratio (ACR), including patients with normoalbuminuria, patients with microalbuminuria, and patients with macroalbuminuria. This cross-sectional study reveals (when the ACR > 30 mg/day was used as the standard for detection of nephropathy), there was a significant difference between groups of diabetic patients (normoalbuminuria, microalbuminuria and macroalbuminuria) with serum cyclophilin A. Receiver operating characteristic (ROC) curves revealed that the best cut-off value of cyclophilin A to differentiate diabetic patients with nephropathy from diabetic patients without nephropathy was ≥ 20.63 ng/ml with 92% sensitivity, 92% specificity and area under the curve (AUC) = 0.978. In conclusion, the serum cyclophilin A can be considered as a good predictor for the diagnosis of DN. [ABSTRACT FROM AUTHOR]

Novel coronavirus disease 2019 (COVID-19) has emerged as a global pandemic with far-reaching societal impact. Here we demonstrate that Pin1 is a key cellular molecule necessary for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) propagation. In this study, siRNA-mediated silencing of Pin1 expression markedly suppressed the proliferation of SARS-CoV-2 in VeroE6/TMPRSS2 cells. In addition, several recently generated Pin1 inhibitors showed strong inhibitory effects on SARS-CoV-2 proliferation, measured by both viral mRNA and protein synthesis, and alleviated the cytopathic effect (CPE) on VeroE6/TMPRSS2 cells. One compound, termed H-77, was found to block SARS-CoV-2 proliferation at an EC50 below 5 μM regardless of whether it was added to the culture medium prior to or after SARS-CoV-2 infection. The inhibition of viral N protein mRNA synthesis by H-77 implies that the molecular mechanism underlying SARS-CoV-2 inhibition is likely to be associated with viral gene transcription or earlier steps. Another Pin1 inhibitor, all-trans retinoic acid (ATRA)—a commercially available drug used to treat acute promyelocytic leukemia (APL) and which both activates the retinoic acid receptor and inhibits the activity of Pin1—similarly reduced the proliferation of SARS-CoV-2. Taken together, the results indicate that Pin1 inhibitors could serve as potential therapeutic agents for COVID-19. [ABSTRACT FROM AUTHOR]

A genome-wide association study (GWAS) was conducted using six different multi-locus GWAS models and 35K SNP array to demarcate genomic regions underlying reproductive stage salinity tolerance. Marker-trait association analysis was performed for salt tolerance indices (STI) of 11 morpho-physiological traits, and the actual concentrations of Na+ and K+, and the Na+/K+ ratio in flag leaf. A total of 293 significantly associated quantitative trait nucleotides (QTNs) for 14 morpho-physiological traits were identified. Of these 293 QTNs, 12 major QTNs with R2 ≥ 10.0% were detected in three or more GWAS models. Novel major QTNs were identified for plant height, number of effective tillers, biomass, grain yield, thousand grain weight, Na+ and K+ content, and the Na+/K+ ratio in flag leaf. Moreover, 48 candidate genes were identified from the associated genomic regions. The QTNs identified in this study could potentially be targeted for improving salinity tolerance in wheat. • Fifty consistent genomic regions were demarcated for 13 morpho-physiological traits using six multi-locus GWAS models. • Four highly consistent QTNs/genomic regions, one each for flag leaf Na+ content (Q.Na-6AL), flag leaf K+ content (Q.K-1AS), above ground biomass (Q.STI-BM-1BL) and Na+/K+ Ratio (Q.NaK-1BS) were identified. • Wheat chromosome 5B harbored the maximum number of genomic regions for yield contributing traits. • The identified QTN regions contained some important stress responsive candidate genes encoding for cytochrome 450 (CYP) family protein, detoxification protein, auxin responsive protein, MYB family transcription factor and peptidylprolyl isomerase (PPI). • ISIS EM-BLASSO is the most efficient model for multi-trait mapping of reproductive stage salinity tolerance. [ABSTRACT FROM AUTHOR]

In vitro generation of red blood cells has the potential to circumvent shortfalls in the global demand for blood for transfusion applications. However, cell differentiation and proliferation are often regulated by precise changes in gene expression, but the underlying mechanisms and molecular changes remain unclear. Quantitative reverse transcription–polymerase chain reaction (qRT-PCR) can be used to evaluate multiple target genes. To make the results more reliable, suitable reference genes should be used to calibrate the error associated with qRT-PCR. In this study, we utilized bioinformatics to screen 3 novel candidate reference genes (calcium and integrin binding family member 2 [CIB2], olfactory receptor family 8 subfamily B member 8 [OR8B8], and zinc finger protein 425 [ZNF425]) along with eight traditional reference genes (glyceraldehyde-3-phosphate dehydrogenase [GAPDH], β-actin [ACTB], 18S RNA, β2-microglobulin [β2-MG], peptidylprolyl isomerase A [PPIA], TATA box-binding protein [TBP], hydroxymethylbilane synthase [HMBS], and hypoxanthine phosphoribosyltransferase 1 [HPRT1]). Two software algorithms (geNorm and NormFinder) were used to evaluate the stability of expression of the 11 genes at different stages of erythrocyte development. Comprehensive analysis showed that expression of GAPDH and TBP was the most stable, whereas ZNF425 and OR8B8 were the least suitable candidate genes. These results suggest that appropriate reference genes should be selected before performing gene expression analysis during erythroid differentiation and that GAPDH and TBP are suitable reference genes for gene expression studies on erythropoiesis. [ABSTRACT FROM AUTHOR]

Gram-negative bacteria are normally resistant to the antibiotic vancomycin (VAN), which cannot significantly penetrate the outer membrane. We used Escherichia coli mutants that are partially sensitive to VAN to study synergies between VAN and 10 other antibiotics representing six different functional categories. We detected strong synergies with VAN and nitrofurantoin (NTR) and with VAN and trimethoprim (TMP) and moderate synergies with other drugs, such as aminoglycosides. These synergies are powerful enough to show the activity of VAN against wild-type E. coli at concentrations of VAN as low as 6.25 μg/ml. This suggests that a very small percentage of exogenous VAN does enter E. coli but normally has insignificant effects on growth inhibition or cell killing. We used the results of pairwise interactions with VAN and the other 10 antibiotics tested to place VAN into a functional category of its own, as previously defined by Yeh et al. (P. Yeh, A. I. Tschumi, and R. Kishony, Nat Genet 28:489-494, 2006, http://dx.doi.org/10.1038/ng1755).

The skeletal muscle is a pivotal organ involved in the regulation of both energy metabolism and exercise capacity. There is no doubt that exercise contributes to a healthy life through the consumption of excessive energy or the release of myokines. Skeletal muscles exhibit insulin sensitivity and can rapidly uptake blood glucose. In addition, they can undergo non-shivering thermogenesis through actions of both the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) and small peptide, sarcolipin, resulting in systemic energy metabolism. Accordingly, the maintenance of skeletal muscles is important for both metabolism and exercise. Prolyl isomerase Pin1 is an enzyme that converts the cis -trans form of proline residues and controls substrate function. We have previously reported that Pin1 plays important roles in insulin release, thermogenesis, and lipolysis. However, the roles of Pin1 in skeletal muscles remains unknown. To clarify this issue, we generated skeletal muscle-specific Pin1 knockout mice. Pin1 deficiency had no effects on muscle weights, morphology and ratio of fiber types. However, they showed exacerbated obesity or insulin resistance when fed with a high-fat diet. They also showed a lower ability to exercise than wild type mice did. We also found that Pin1 interacted with SERCA and elevated its activity, resulting in the upregulation of oxygen consumption. Overall, our study reveals that Pin1 in skeletal muscles contributes to both systemic energy metabolism and exercise capacity. • Pin1 has no effects on muscle weights or the ratio of fiber types. • Pin1 deficiency in skeletal muscles worsens obesity and impairs exercise capacity. • Pin1 interacts with SERCA and upregulates its activity. • Pin1 in skeletal muscles enhances oxygen consumption. [ABSTRACT FROM AUTHOR]

Peptidyl‐prolyl cis‐trans isomerase C (Ppic) is expressed in several bone marrow (BM) hematopoietic progenitors and in T‐cell precursors. Since the expression profile of Ppic in the hematoimmune system was suggestive that it could play a role in hematopoiesis and/or T lymphocyte differentiation, we sought to test that hypothesis in vivo. Specifically, we generated a Ppic‐deficient mouse model by targeting the endogenous locus by CRISPR/Cas9 and tested the requirement of Ppic in hematopoiesis. Several immune cell lineages covering BM progenitors, lymphocyte precursors, as well as mature cells at the periphery were analyzed. While most lineages were unaffected, invariant NKT (iNKT) cells were reduced in percentage and absolute cell numbers in the Ppic‐deficient thymus. This affected the most mature stages in the thymus, S2 and S3, and the phenotype was maintained at the periphery. Additionally, immature transitional T1 and T2 B lymphocytes were increased in the Ppic‐deficient spleen, but the phenotype was lost in mature B lymphocytes. In sum, our data show that Ppic is dispensable for myeloid cells, platelets, erythrocytes, αβ, and γδ T lymphocytes in vivo in the steady state, while being involved in B‐ and iNKT cell differentiation. [ABSTRACT FROM AUTHOR]

The GPN proteins are a conserved family of GTP-binding proteins that are involved in the assembly and subsequent import of RNA polymerase II and III. In this study, we sought to ascertain the specificity of yeast GPN2 for RNA polymerases by screening the localization of a collection of 1350 GFP-tagged nuclear proteins in WT or GPN2 mutant cells. We found that the strongest mislocalization occurred for RNA polymerase II and III subunits and only a handful of other RNAPII associated proteins were altered in GPN2 mutant cells. Our screen identified Ess1, an Rpb1 C-terminal domain (CTD) prolyl isomerase, as mislocalized in GPN2 mutants. Building on this observation we tested for effects of mutations in other factors which regulate Rpb1-CTD phosphorylation status. This uncovered significant changes in nuclear-cytoplasmic distribution of Rpb1-GFP in strains with disrupted RNA polymerase CTD kinases or phosphatases. Overall, this screen shows the exquisite specificity of GPN2 for RNA polymerase transport, and reveals a previously unappreciated role for CTD modification in RNAPII nuclear localization. [ABSTRACT FROM AUTHOR]

Protein kinases are important regulators in cellular signal transduction. As one major type of Hsp90 client, protein kinases rely on the ATP-dependent molecular chaperone Hsp90, which maintains their structure and supports their activation. Depending on client type, Hsp90 interacts with different cofactors. Here we report that besides the kinase-specific cofactor Cdc37 large PPIases of the Fkbp-type strongly bind to kinase•Hsp90•Cdc37 complexes. We evaluate the nucleotide regulation of these assemblies and identify prominent interaction sites in this quaternary complex. The synergistic interaction between the participating proteins and the conserved nature of the interaction suggests functions of the large PPIases Fkbp51/Fkbp52 and their nematode homolog FKB-6 as contributing factors to the kinase cycle of the Hsp90 machinery. [ABSTRACT FROM AUTHOR]

Amyotrophic lateral sclerosis and frontotemporal dementia are two neurodegenerative diseases with overlapping clinical features and the pathological hallmark of cytoplasmic deposits of misfolded proteins. The most frequent cause of familial forms of these diseases is a hexanucleotide repeat expansion in the non-coding region of the C9ORF72 gene that is translated into dipeptide repeat polymers. Here we show that proline/arginine repeat polymers derail protein folding by sequestering molecular chaperones. We demonstrate that proline/arginine repeat polymers inhibit the folding catalyst activity of PPIA, an abundant molecular chaperone and prolyl isomerase in the brain that is altered in amyotrophic lateral sclerosis. NMR spectroscopy reveals that proline/arginine repeat polymers bind to the active site of PPIA. X-ray crystallography determines the atomic structure of a proline/arginine repeat polymer in complex with the prolyl isomerase and defines the molecular basis for the specificity of disease-associated proline/arginine polymer interactions. The combined data establish a toxic mechanism that is specific for proline/arginine dipeptide repeat polymers and leads to derailed protein homeostasis in C9orf72-associated neurodegenerative diseases. The most frequent cause of familial Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD) are hexanucleotide repeat expansions in the non-coding region of the C9ORF72 gene that are translated into five dipeptide repeat (DPR) proteins. Here, the authors show that proline/arginine (PR) DPRs inhibit the prolyl isomerase PPIA and reveal the molecular mechanism of the impaired protein folding activity of PPIA by performing NMR measurements and determining a PR DPR bound PPIA crystal structure. [ABSTRACT FROM AUTHOR]

Synaptic plasticity induced by cocaine and other drugs underlies addiction. Here we elucidate molecular events at synapses that cause this plasticity and the resulting behavioral response to cocaine in mice. In response to D1-dopamine-receptor signaling that is induced by drug administration, the glutamate-receptor protein metabotropic glutamate receptor 5 (mGluR5) is phosphorylated by microtubule-associated protein kinase (MAPK), which we show potentiates Pin1-mediated prolyl-isomerization of mGluR5 in instances where the product of an activity-dependent gene, Homer1a, is present to enable Pin1-mGluR5 interaction. These biochemical events potentiate N-methyl-D-aspartate receptor (NMDAR)-mediated currents that underlie synaptic plasticity and cocaine-evoked motor sensitization as tested in mice with relevant mutations. The findings elucidate how a coincidence of signals from the nucleus and the synapse can render mGluR5 accessible to activation with consequences for drug-induced dopamine responses and point to depotentiation at corticostriatal synapses as a possible therapeutic target for treating addiction.

Reverse transcription quantitative real-time PCR is the most commonly used method to detect gene expression levels. In experiments, it is often necessary to correct and standardize the expression level of target genes with reference genes. Therefore, it is very important to select stable reference genes to obtain accurate quantitative results. Although application examples of reference genes in mammals have been reported, no studies have investigated the use of reference genes in studying the growth and development of adipose tissue and the proliferation and differentiation of preadipocytes in chickens. In this study, GeNorm, a reference gene stability statistical algorithm, was used to analyze the expression stability of 14 candidate reference genes in the abdominal adipose tissue of broilers at 1, 4, and 7 weeks of age, the proliferation and differentiation of primary preadipocytes, as well as directly isolated preadipocytes and mature adipocytes. The results showed that the expression of the TATA box binding protein (TBP) and hydroxymethylbilane synthase (HMBS) genes was most stable during the growth and development of abdominal adipose tissue of broilers, the expression of the peptidylprolyl isomerase A (PPIA) and HMBS genes was most stable during the proliferation of primary preadipocytes, the expression of the TBP and RPL13 genes was most stable during the differentiation of primary preadipocytes, and the expression of the TBP and HMBS genes was most stable in directly isolated preadipocytes and mature adipocytes. These results provide reference bases for accurately detecting the mRNA expression of functional genes in adipose tissue and adipocytes of chickens. [ABSTRACT FROM AUTHOR]

Accurate gene transcription in eukaryotes depends on isomerization of serine-proline bonds within the carboxy-terminal domain (CTD) of RNA polymerase II. Isomerization is part of the "CTD code" that regulates recruitment of proteins required for transcription and co-transcriptional RNA processing. Saccharomyces cerevisiae Ess1 and its human ortholog, Pin1, are prolyl isomerases that engage the long heptad repeat (YSPTSPS)26 of the CTD by an unknown mechanism. Here, we used an integrative structural approach to decipher Ess1 interactions with the CTD. Ess1 has a rigid linker between its WW and catalytic domains that enforces a distance constraint for bivalent interaction with the ends of long CTD substrates (≥4–5 heptad repeats). Our binding results suggest that the Ess1 WW domain anchors the proximal end of the CTD substrate during isomerization, and that linker divergence may underlie evolution of substrate specificity. Namitz, Zheng et al. identify a bivalent interaction by the yeast Ess1 with CTD peptides of RNA polymerase II. Their results suggest an anchored mechanism of isomerization, and raise the possibility of eukaryotic parvulin-class prolyl isomerases gaining a broader substrate specificity during evolution, by acquiring a flexible linker that generates a more dynamic binding mode. [ABSTRACT FROM AUTHOR]

The burden of hepatocellular carcinoma (HCC) worldwide is increasing over time, while the underlying molecular mechanism of HCC development is still under exploration. Pseudogenes are classified as a special type of long non-coding RNAs (lncRNAs), and they played a vital role in regulating tumor-associated gene expression. Here, we report that a pseudogene peptidylprolyl isomerase A pseudogene 22 (PPIAP22) and its parental gene peptidylprolyl isomerase A (PPIA) were upregulated in HCC and were associated with the clinical outcomes of HCC. Further investigation revealed that PPIAP22 might upregulate the expression of PPIA through sponging microRNA (miR)-197-3p, behaving as competing endogenous RNA (ceRNA). PPIA could participate in the development of HCC by regulating mRNA metabolic process and tumor immunity based on the functional enrichment analysis. We also found a strong correlation between the expression levels of PPIA and the immune cell infiltration or the expression of chemokines, especially macrophage, C-C motif chemokine ligand 15 (CCL15), and C-X-C motif chemokine ligand 12 (CXCL12). Our findings demonstrate that the PPIAP22/miR-197-3p/PPIA axis plays a vital role in the progression of HCC by increasing the malignancy of tumor cells and regulating the immune cell infiltration, especially macrophage, through CCL15-CCR1 or CXCL12-CXCR4/CXCR7 pathways. [ABSTRACT FROM AUTHOR]

Molecular chaperones are central to the maintenance of proteostasis in living cells. A key member of this protein family is trigger factor (TF), which acts throughout the protein life cycle and has a ubiquitous role as the first chaperone encountered by proteins during synthesis. However, our understanding of how TF achieves favorable interactions with such a diverse substrate base remains limited. Here, we use microfluidics to reveal the thermodynamic determinants of this process. We find that TF binding to empty 70S ribosomes is enthalpy-driven, with micromolar affinity, while nanomolar affinity is achieved through a favorable entropic contribution for both intrinsically disordered and folding-competent nascent chains. These findings suggest a general mechanism for cotranslational TF function, which relies on occupation of the exposed TF-substrate binding groove rather than specific complementarity between chaperone and nascent chain. These insights add to our wider understanding of how proteins can achieve broad substrate specificity.

Protein folding is assisted by molecular chaperones that bind nascent polypeptides during mRNA translation. Several structurally distinct classes of chaperones promote de novo folding, suggesting that their activities are coordinated at the ribosome. We used biochemical reconstitution and structural proteomics to explore the molecular basis for cotranslational chaperone action in bacteria. We found that chaperone binding is disfavored close to the ribosome, allowing folding to precede chaperone recruitment. Trigger factor recognizes compact folding intermediates that expose an extensive unfolded surface, and dictates DnaJ access to nascent chains. DnaJ uses a large surface to bind structurally diverse intermediates and recruits DnaK to sequence-diverse solvent-accessible sites. Neither Trigger factor, DnaJ, nor DnaK destabilize cotranslational folding intermediates. Instead, the chaperones collaborate to protect incipient structure in the nascent polypeptide well beyond the ribosome exit tunnel. Our findings show how the chaperone network selects and modulates cotranslational folding intermediates., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Protein-protein interactions are often mediated by flexible loops that experience conformational dynamics on the microsecond to millisecond time scales. NMR relaxation studies can map these dynamics. However, defining the network of inter-converting conformers that underlie the relaxation data remains generally challenging. Here, we combine NMR relaxation experiments with simulation to visualize networks of inter-converting conformers. We demonstrate our approach with the apo Pin1-WW domain, for which NMR has revealed conformational dynamics of a flexible loop in the millisecond range. We sample and cluster the free energy landscape using Markov State Models (MSM) with major and minor exchange states with high correlation with the NMR relaxation data and low NOE violations. These MSM are hierarchical ensembles of slowly interconverting, metastable macrostates and rapidly interconverting microstates. We found a low population state that consists primarily of holo-like conformations and is a "hub" visited by most pathways between macrostates. These results suggest that conformational equilibria between holo-like and alternative conformers pre-exist in the intrinsic dynamics of apo Pin1-WW. Analysis using MutInf, a mutual information method for quantifying correlated motions, reveals that WW dynamics not only play a role in substrate recognition, but also may help couple the substrate binding site on the WW domain to the one on the catalytic domain. Our work represents an important step towards building networks of inter-converting conformational states and is generally applicable.

Pin1 is the only known peptidyl-prolyl cis-trans isomerase (PPIase) that can specifically recognize and isomerize the phosphorylated Serine/Threonine-Proline (pSer/Thr-Pro) motif, change the conformation of proteins through protein phosphorylation, thus regulate various cellular processes in the body. Pin1 plays an important role in cancer, Alzheimer's disease, and autoimmune diseases. However, the specific mechanism of Pin1 regulation in LPS-induced septic shock is unclear. Here, we found that lack of Pin1 reduced shock mortality and organ damage in mice, and NLRP3 inflammasome activation also was reduced in this process. We further confirmed that Pin1 can affect the expression of NLRP3, ASC, Caspase1, and this process can be regulated through the p38 MAPK pathway. We analyzed that p38 MAPK signaling pathway was highly expressed in septic shock and showed a positive correlation with Pin1 in the Gene Expression Omnibus database. We found that Pin1 could affect the phosphorylation of p38 MAPK, have no obvious difference in extracellular signal-regulated kinases (ERK) and Jun-amino-terminal kinase (JNK) signaling. We further found that Pin1 and p-p38 MAPK interacted, but not directly. In addition, Pin1 deficiency inhibited the cleavage of gasdermin D (GSDMD) and promoted the death of macrophages with LPS treatment, and reduced secretion of inflammatory cytokines including IL-1β and IL-18. In general, our results suggest that Pin1 regulates the NLRP3 inflammasome activation by p38 MAPK signaling pathway in macrophages. Thus, Pin1 may be a potential target for the treatment of inflammatory diseases such as septic shock. [ABSTRACT FROM AUTHOR]

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. Author summary: Metal ions are essential for the viability of all living organisms. Indeed, more than one-third of all proteins need metal cofactors for function. Intracellular metal concentrations require tight control as non-physiological amounts are very toxic. In particular, nickel plays a unique role in Helicobacter pylori, a bacterial pathogen that colonizes the stomach of about half of the human population worldwide and is associated with the development of gastric cancer. Nickel is essential for H. pylori as it is the cofactor of urease, an enzyme indispensable for resistance to the gastric acidity of the stomach and thus for in vivo colonization. To import nickel despite its scarcity in the human body, H. pylori requires efficient uptake mechanisms. Till now, control of nickel uptake was only reported to rely on transcriptional regulators. In the present study, we uncovered a novel mechanism of regulation of nickel acquisition. SlyD, a multifunctional enzyme was found to control, by direct protein interaction, the activity of an essential nickel uptake system in H. pylori. We revealed that the SlyD chaperone activity is mandatory for the active conformation and thus functionality of the nickel permease. [ABSTRACT FROM AUTHOR]

Drought stress is the most important limiting factor of crop cultivation around the world and stress conditions will modulate the expression of many genes that play a significant role in the plant growth and development and to stress adaptation. The apoplast is a dynamic environment subject and considered as a 'key information bridge' between the environment and cells. In the present study we aimed to measure the changes in the expression levels of genes by RT-qPCR studies.Analysis shown the down-regulation of Peptidylprolyl isomerase and Pin II type protease inhibitor and there observed the huge increase in the expression of Pectinesterase and Alpha L fucosidase under treated conditions. These changes in the expression level of proteins under stress conditions implicate their possible role in stress tolerance and would prove to be useful for protein phenotyping. [ABSTRACT FROM AUTHOR]

PIN1, the peptidyl-prolyl isomerase (PPIase), is an enzyme that changes the conformation of phosphoproteins. The conformational change induced by PIN1 alters the function and stability of the target proteins. PIN1 is overexpressed in many different types of malignancies, including breast, lung, cervical, brain and colorectal tumors. PIN1 overexpression has been associated with activation of multiple oncogenic signaling pathways during tumor development. Hypoxia-inducible factor 2α (HIF-2α), a transcription factor activated in hypoxia, plays a role in erythropoiesis, glycolysis, tissue invasion, metastasis and angiogenesis. In this study, we found the direct interaction between HIF-2α and PIN1 in colorectal cancer HCT116 cells. Notably, serine 16 and lysine 63 residues of PIN1 were critical for its interaction with HIF-2α. When PIN1 protein was silenced by transient transfection of PIN1 short interfering RNA, the expression of HIF-2α was attenuated under a hypoxic condition. Moreover, genetic and pharmacologic inhibition of PIN1 abrogated the expression of vascular endothelial growth factor and angiogenesis. The cycloheximide chase experiment revealed the stabilization of HIF-2α by PIN1. Both WW and PPIase domains of PIN1 appear to be critical for its interaction with HIF-2α. Image 1 • PIN1 binds directly to HIF-2α in colorectal cancer HCT116 cells. • PIN1 interaction with HIF-2α stabilizes this transcription factor. • K63A mutation in the catalytically active PPIase domain and S16A mutation in the WW domain of PIN1 abolished its interaction with HIF-2α. • Genetic or pharmacologic inhibition of PIN1 abrogated the angiogenesis. [ABSTRACT FROM AUTHOR]

The functional mechanisms of multidomain proteins often exploit interdomain interactions, or "cross-talk." An example is human Pin1, an essential mitotic regulator consisting of a Trp- Trp (WW) domain flexibly tethered to a peptidyl-prolyl isomerase (PPIase) domain, resulting in interdomain interactions important for Pin1 function. Substrate binding to the WW domain alters its transient contacts with the PPIase domain via means that are only partially understood. Accordingly, we have investigated Pin1 interdomain interactions using NMR paramagnetic relaxation enhancement (PRE) and molecular dynamics (MD) simulations. The PREs show that apo-Pin1 samples interdomain contacts beyond the range suggested by previous structural studies. They further show that substrate binding to the WW domain simultaneously alters interdomain separation and the internal conformation of the WW domain. A 4.5-ms all-atom MD simulation of apo-Pin1 suggests that the fluctuations of interdomain distances are correlated with fluctuations of WW domain interresidue contacts involved in substrate binding. Thus, the interdomain/WW domain conformations sampled by apo-Pin1 may already include a range of conformations appropriate for binding Pin1's numerous substrates. The proposed coupling between intra-/interdomain conformational fluctuations is a consequence of the dynamic modular architecture of Pin1. Such modular architecture is common among cell-cycle proteins; thus, the WW-PPIase domain cross-talk mechanisms of Pin1 may be relevant for their mechanisms as well. [ABSTRACT FROM AUTHOR]

Neoporphyra haitanensis is an economically important red seaweed that inhabits upper intertidal zones. The thallus tolerates extreme fluctuating environmental stresses (e.g., surviving more than 80% water loss during low tides). To elucidate the global molecular responses relevant to this outstanding desiccation tolerance, a quantitative proteomics analysis of N. haitanensis under different desiccation treatments as well as rehydration was performed. According to the clustering of expression patterns and the functional interpretation of the 483 significantly differentially expressed proteins, a three‐stage cellular response to desiccation stress and subsequent rehydration was proposed. Stage I: at the beginning of water loss, multiple signal transduction pathways were triggered including lipid signaling, protein phosphorylation cascades, and histone acetylation controlling acetate biosynthesis to further modulate downstream hormone signaling. Protein protection by peptidyl‐prolyl isomerase and ROS scavenging systems were also immediately switched on. Stage II: with the aggravation of stress, increases in antioxidant systems, the accumulation of LEA proteins, and the temporary biosynthesis of branched starch were observed. Multiple enzymes involved in redox homeostasis, including peroxiredoxin, thioredoxin, ascorbate peroxidase, superoxide dismutase, glutathione peroxidase, and glutathione reductase, were hypothesized to function in specific cellular compartments. Stage III: when the desiccated thalli had rehydrated for 30 mins, photosynthesis and carbon fixation were recovered, and antioxidant activities and protein structure protection were maintained at a high level. This work increases the understanding of the molecular responses to environmental stresses via a proteomic approach in red seaweeds and paves the way for further functional studies and genetic engineering. [ABSTRACT FROM AUTHOR]

Post‐transcriptional regulation of ATP‐binding cassette (ABC) proteins has been so far shown to encompass protein phosphorylation, maturation, and ubiquitination. Yet, recent accumulating evidence implicates FK506‐binding proteins (FKBPs), a type of peptidylprolyl cis–trans isomerase (PPIase) proteins, in ABC transporter regulation. In this perspective article, we summarize current knowledge on ABC transporter regulation by FKBPs, which seems to be conserved over kingdoms and ABC subfamilies. We uncover striking functional similarities but also differences between regulatory FKBP‐ABC modules in plants and mammals. We dissect a PPIase‐ and HSP90‐dependent and independent impact of FKBPs on ABC biogenesis and transport activity. We propose and discuss a putative new mode of transient ABC transporter regulation by cis–trans isomerization of X‐prolyl bonds. [ABSTRACT FROM AUTHOR]

Objective: Quantitative polymerase chain reaction (qPCR) has been extensively used in the field of mesenchymal stem cell (MSC) research to elucidate their characteristics and clinical potential by normalization of target genes against reference genes (RGs), which are believed to be stably expressed irrespective of various experimental conditions. However, the expression of RGs is also variable depending on the experimental conditions, which may lead to false or contradictory conclusions upon normalization. Due to the current lack of information for a clear list of stable RGs in bovine MSCs, we conducted this study to identify suitable RGs in bovine MSCs. Methods: The cycle threshold values of ten traditionally used RGs (18S ribosomal RNA [18S], beta-2-microglobulin [B2M], H2A histone family, member Z [H2A], peptidylprolyl isomerase A [PPIA], ribosomal protein 4 [RPL4], succinate dehydrogenase complex, subunit A [SDHA], beta actin [ACTB], glyceraldehyde-3-phosphate dehydrogenase [GAPDH], TATA box binding protein [TBP], and hypoxanthine phosphoribosyltrasnfrase1 [HPRT1]) in bovine bone marrow-derived MSCs (bBMMSCs) were validated for their stabilities using three types of RG evaluation algorithms (geNorm, Normfinder, and Bestkeeper). The effect of validated RGs was then verified by normalization of lineage-specific genes (fatty acid binding protein 4 [FABP4] and osteonectin [ON]) expressions during differentiations of bBMMSCs or POU class 5 homeobox 1 (OCT4) expression between bBMMSCs and dermal skins. Results: Based on the results obtained for the three most stable RGs from geNorm (TBP, RPL4, and H2A), Normfinder (TBP, RPL4, and SDHA), and Bestkeeper (TBP, RPL4, and SDHA), it was comprehensively determined that TBP and RPL4 were the most stable RGs in bBMMSCs. However, traditional RGs were suggested to be the least stable (18S) or moderately stable (GAPDH and ACTB) in bBMMSCs. Normalization of FABP4 or ON against TBP, RPL4, and 18S presented significant differences during differentiation of bBMMSCs. However, although significantly low expression of OCT4 was detected in dermal skins compared to that in bBMMSCs when TBP and RPL4 were used in normalization, normalization against 18S exhibited no significance. Conclusion: This study proposes that TBP and RPL4 were suitable as stable RGs for qPCR study in bovine MSCs. [ABSTRACT FROM AUTHOR]

Caldicellulosiruptor bescii secretes a large number of complementary multifunctional enzymes with unique activities for biomass deconstruction. The most abundant in the C. bescii secretome are found in a unique gene cluster containing a glycosyl transferase (GT39) and a putative peptidyl prolyl cis-trans isomerase. Deletion of the glycosyl transferase in this cluster resulted is loss of detectable protein glycosylation in C. bescii and its activity has been shown to be responsible for the glycosylation of the proline31 threonine rich linkers found in many of the multifunctional cellulases. The presence of a putative peptidyl prolyl cis-trans isomerase within this gene cluster suggested that it might also play a role in cellulase modification. In this work, we identify this gene as a putative prsA prolyl cis-trans isomerase. Deletion of prsA2 leads to the inability of C. bescii to grow on insoluble substrates such as Avicel, the model cellulose substrate, while exhibiting no differences in phenotype with the wild type strain on soluble substrates. Finally, we provide evidence that the prsA2 gene is likely needed to increase solubility of multifunctional cellulases and that this unique gene cluster was likely acquired by members of the Caldicellulosiruptor genus with a group of genes to optimize the production and activity of multifunctional cellulases. [ABSTRACT FROM AUTHOR]

The plant hormone auxin must be transported throughout plants in a cell-to-cell manner to affect its various physiological functions. ABCB transporters are critical for this polar auxin distribution, but the regulatory mechanisms controlling their function is not fully understood. The auxin transport activity of ABCB1 was suggested to be regulated by a physical interaction with FKBP42/Twisted Dwarf1 (TWD1), a peptidylprolyl cis-trans isomerase (PPIase), but all attempts to demonstrate such a PPIase activity by TWD1 have failed so far. By using a structure-based approach, we identified several surface-exposed proline residues in the nucleotide binding domain and linker of Arabidopsis ABCB1, mutations of which do not alter ABCB1 protein stability or location but do affect its transport activity. P1008 is part of a conserved signature D/E-P motif that seems to be specific for auxin-transporting ABCBs, which we now refer to as ATAs. Mutation of the acidic residue also abolishes auxin transport activity by ABCB1. All higher plant ABCBs for which auxin transport has been conclusively proven carry this conserved motif, underlining its predictive potential. Introduction of this D/E-P motif into malate importer, ABCB14, increases both its malate and its background auxin transport activity, suggesting that this motif has an impact on transport capacity. The D/E-P1008 motif is also important for ABCB1-TWD1 interactions and activation of ABCB1-mediated auxin transport by TWD1. In summary, our data imply a new function for TWD1 acting as a putative activator of ABCB-mediated auxin transport by cis-trans isomerization of peptidyl-prolyl bonds. [ABSTRACT FROM AUTHOR]

Death‐associated protein kinase 1 (DAPK1) is upregulated in the brains of human Alzheimer's disease (AD) patients compared with normal subjects, and aberrant DAPK1 regulation is implicated in the development of AD. However, little is known about whether and how DAPK1 function is regulated in AD. Here, we identified melatonin as a critical regulator of DAPK1 levels and function. Melatonin significantly decreases DAPK1 expression in a post‐transcriptional manner in neuronal cell lines and mouse primary cortical neurons. Moreover, melatonin directly binds to DAPK1 and promotes its ubiquitination, resulting in increased DAPK1 protein degradation through a proteasome‐dependent pathway. Furthermore, in tau‐overexpressing mouse brain slices, melatonin treatment and the inhibition of DAPK1 kinase activity synergistically decrease tau phosphorylation at multiple sites related to AD. In addition, melatonin and DAPK1 inhibitor dramatically accelerate neurite outgrowth and increase the assembly of microtubules. Mechanistically, melatonin‐mediated DAPK1 degradation increases the activity of Pin1, a prolyl isomerase known to play a protective role against tau hyperphosphorylation and tau‐related pathologies. Finally, elevated DAPK1 expression shows a strong correlation with the decrease in melatonin levels in human AD brains. Combined, these results suggest that DAPK1 regulation by melatonin is a novel mechanism that controls tau phosphorylation and function and offers new therapeutic options for treating human AD. [ABSTRACT FROM AUTHOR]

Cervical carcinoma is the most common gynecological cancer in women worldwide. Emerging evidence has shown that long non-coding RNAs (lncRNAs) participate in multiple biological processes of cervical carcinoma tumorigenesis. We aimed to investigate the function of a novel lncRNA RP11-284F21.9 in cervical carcinoma. We found that RP11-284F21.9 was down-regulated in cervical carcinoma tissues and cell lines. Overexpression of RP11-284F21.9 inhibits proliferation, invasion and migration of cervical carcinoma cells in vitro. Further, we identified that RP11-284F21.9 directly interacted with miR-769-3p and functioned as the miR-769-3p sponge. Mechanistically, we showed that miR-769-3p regulated peptidylprolyl isomerase domain and WD repeat-containing protein1 (PPWD1) expression by targeting PPWD1 3-UTR. Furthermore, xenograft tumor model revealed that overexpression of RP11-284F21.9 inhibited tumor growth of cervical carcinoma in vivo. Taken together, our results demonstrate that RP11-284F21.9 functions as tumor suppressor and regulates PPWD1 expression through competitively binding to miR-769-3p in cervical carcinoma, suggesting that RP11-284F21.9/miR-769-3p/PPWD1 axis could serve as a promising prognostic biomarker and therapeutic target for cervical carcinoma. [ABSTRACT FROM AUTHOR]

aimei Li,1 Fuqiang Wang,1 Yufen Yuan11Department of Pathology, Anyang Tumor Hospital, The Fourth Affiliated Hospital of Henan University of Science and Technology, Anyang 455000, People's Republic of China; 2NHC Key Laboratory of Biotechnology of Antibiotics, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100032, People's Republic of ChinaCorrespondence: Yufen Yuan Tel +86 18810845563Email yuanyufen2008@163.com Introduction: Diffuse large B cell lymphoma (DLBCL) is a highly heterogeneous type of non-Hodgkin lymphoma with many molecular subtypes that can be distinguished by gene expression profiling (GEP). However, the pathogenesis of DLBCL is still unclear. Materials and Methods: The expression levels of the prolyl isomerase PIN-1 and other related proteins were determined in 73 primary DLBCL patient samples and cell lines by Western blotting (WB) and immunohistochemical (IHC) staining. Cell cycle and apoptosis were evaluated by flow cytometry. Lymphoma cell viability was detected by CCK-8 proliferation assay. Results: High levels of PIN-1 expression were detected in 55% of germinal center B cell (GCB) DLBCL patient samples, whereas such abnormal expression levels were found in only 11% of non-GCB DLBCL patient samples. PIN-1 expression was positively associated with activation of the oncogenic phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway in both GCB DLBCL cell lines and primary patient samples. Depletion of PIN-1 was cytotoxic to GCB DLBCL model cell lines because it led to inhibition of the PI3K/AKT signaling pathway, revealing a GCB DLBCL subgroup that is dependent on this pathway. A PI3K inhibitor was selectively toxic to GCB DLBCL lines expressing high levels of PIN-1. Conclusion: Our study used PIN-1 to identify a new subgroup of GCB DLBCL associated with the PI3K/AKT signaling pathway, and our findings reveal that inhibition of PI3K is a promising therapeutic approach for GCB DLBCL. [ABSTRACT FROM AUTHOR]

Background/objective Periodontal ligament fibroblasts (PDLF) play an important mediating role in orthodontic tooth movement expressing various cytokines, when exposed to compressive or tensile strain. Here, we present a simplified and easy-to-handle, but reliable and valid method for simulating static isotropic tensile strain in vitro using spherical silicone cap stamps. Furthermore, we identify appropriate reference genes for data normalization in real-time quantitative polymerase chain reaction (RT-qPCR) experiments on PDLF subjected to tensile strain. Materials and methods PDLF were cultivated on flexible bioflex membranes and exposed to static isotropic tensile strain of different magnitudes and timeframes. We determined cell number, cytotoxicity, and relative expression of proinflammatory genes cyclooxygenase-2 (COX-2) and interleukin-6 (IL-6). For normalization of RT-qPCR data, we tested the stability and validity of nine candidate reference genes with four mathematical algorithms (geNorm, NormFinder, comparative ΔCq, and BestKeeper) and ranked them based on their calculated expression stability. Results We observed no decrease in cell number or cytotoxic effect at any of the applied magnitudes and timeframes of tensile strain. At 16 per cent and 35 per cent tensile strain for 48 hours, we detected a significant increase in COX-2 and decrease in IL-6 gene expression. Highest stability was found for TBP (TATA-box-binding protein) and PPIB (peptidylprolyl isomerase A) in reference gene validation. According to the geNorm algorithm, both genes in conjunction are sufficient for normalization. In contrast to all other candidate genes tested, gene expression normalization of target gene COX-2 to reference genes EEF1A1 , RPL22 , and RNA18S5 indicated no significant upregulation of COX-2 expression. Conclusions A strain magnitude of 16 per cent for 48 hours elicited the most distinct cellular response by PDLF subjected to static tensile isotropic strain by the presented method. TBP and PPIB in conjunction proved to be the most appropriate reference genes to normalize target gene expression in RT-qPCR studies on PDLF subjected to tensile strain. [ABSTRACT FROM AUTHOR]

Background: We present a functional gene association network of the CLIP2 gene, generated by de-novo reconstruction from transcriptomic microarray data. CLIP2 was previously identified as a potential marker for radiation induced papillary thyroid carcinoma (PTC) of young patients in the aftermath of the Chernobyl reactor accident. Considering the rising thyroid cancer incidence rates in western societies, potentially related to medical radiation exposure, the functional characterization of CLIP2 is of relevance and contributes to the knowledge about radiation-induced thyroid malignancies.Methods: We generated a transcriptomic mRNA expression data set from a CLIP2-perturbed thyroid cancer cell line (TPC-1) with induced CLIP2 mRNA overexpression and siRNA knockdown, respectively, followed by gene-association network reconstruction using the partial correlation-based approach GeneNet. Furthermore, we investigated different approaches for prioritizing differentially expressed genes for network reconstruction and compared the resulting networks with existing functional interaction networks from the Reactome, Biogrid and STRING databases. The derived CLIP2 interaction partners were validated on transcript and protein level.Results: The best reconstructed network with regard to selection parameters contained a set of 20 genes in the 1st neighborhood of CLIP2 and suggests involvement of CLIP2 in the biological processes DNA repair/maintenance, chromosomal instability, promotion of proliferation and metastasis. Peptidylprolyl Isomerase Like 3 (PPIL3), previously identified as a potential direct interaction partner of CLIP2, was confirmed in this study by co-expression at the transcript and protein level.Conclusion: In our study we present an optimized preselection approach for genes subjected to gene-association network reconstruction, which was applied to CLIP2 perturbation transcriptome data of a thyroid cancer cell culture model. Our data support the potential carcinogenic role of CLIP2 overexpression in radiation-induced PTC and further suggest potential interaction partners of the gene. [ABSTRACT FROM AUTHOR]

Purpose: To explore the role of FKBP prolyl isomerase 10 (FKBP10) protein in the progression of gastric cancer. Methods: Four independent gastric cancer databases (GSE27342, GSE29272, GSE54129 and TCGA-STAD) were used to identify differentially expressed genes (DEGs). Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was used to identify the abnormally active pathways in patients with gastric cancer. Univariate Cox regression analysis was used to identify genes with stable prognostic value in gastric cancer patients based on three independent gastric cancer databases (GSE15459, GSE62254, TCGA-STAD). Gene set enrichment analysis (GSEA) was used to explore the possible pathways related to FKBP10. The reverse transcription-polymerase chain reaction (RT-PCR) was employed to determine the expression of FKBP10 mRNA in the HGC-27 and MKN-7 cell lines. Adhesion assay was used to detect changes in cell adhesion ability. FKBP10, ITGA1, ITGA2, ITGA5, ITGAV, ITGA6, P-AKT473, P-AKT308, AKT, and β-actin were evaluated by Western blot (WB). Results: We first performed differential expression genes (DEGs) screening of four independent GC databases (GSE27342, GSE29272, GSE54129 and TCGA-STAD). Eighty-nine genes showed consistent up-regulation in GC, the results of pathway analysis showed that they were related to "Focal adhesion". The prognostic value of these 89 genes was tested in three independent GC databases GSE15459, GSE62254 and TCGA-STAD cohort. Finally, 12 genes, in which the expression of FKBP10 was prominently increased in patients with lymph node metastasis (LNM), showed stable prognostic value. The following gene set enrichment analysis (GSEA) also showed that FKBP10 is mainly involved in cell adhesion process, while adhesion experiments confirmed that cell adhesion was down-regulated after silencing FKBP10 in GC cells, and adhesion-related molecules integrin αV and α 6 were down-regulated. Conclusion: FKBP10 may be used as a marker for lymph node metastasis of GC and could be used as a potential target for future treatment of GC. [ABSTRACT FROM AUTHOR]

The function of steroid receptors in the cell depends on the chaperone machinery of Hsp90, as Hsp90 primes steroid receptors for hormone binding and transcriptional activation. Several conserved proteins are known to additionally participate in receptor chaperone assemblies, but the regulation of the process is not understood in detail. Also, it is unknown to what extent the contribution of these cofactors is conserved in other eukaryotes. We here examine the reconstituted C. elegans and human chaperone assemblies. We find that the nematode phosphatase PPH-5 and the prolyl isomerase FKB-6 facilitate the formation of glucocorticoid receptor (GR) complexes with Hsp90. Within these complexes, Hsp90 can perform its closing reaction more efficiently. By combining chemical crosslinking and mass spectrometry, we define contact sites within these assemblies. Compared to the nematode Hsp90 system, the human system shows less cooperative client interaction and a stricter requirement for the co-chaperone p23 to complete the closing reaction of GR·Hsp90·Pp5/Fkbp51/Fkbp52 complexes. In both systems, hormone binding to GR is accelerated by Hsp90 alone and in the presence of its cofactors. Our results show that cooperative complex formation and hormone binding patterns are, in many aspects, conserved between the nematode and human systems. [ABSTRACT FROM AUTHOR]

The periplasmic chaperone SurA plays a key role in outer membrane protein (OMP) biogenesis. E. coli SurA comprises a core domain and two peptidylprolyl isomerase domains (P1 and P2), but its mechanisms of client binding and chaperone function have remained unclear. Here, we use chemical cross-linking, hydrogen-deuterium exchange mass spectrometry, single-molecule FRET and molecular dynamics simulations to map the client binding site(s) on SurA and interrogate the role of conformational dynamics in OMP recognition. We demonstrate that SurA samples an array of conformations in solution in which P2 primarily lies closer to the core/P1 domains than suggested in the SurA crystal structure. OMP binding sites are located primarily in the core domain, and OMP binding results in conformational changes between the core/P1 domains. Together, the results suggest that unfolded OMP substrates bind in a cradle formed between the SurA domains, with structural flexibility between domains assisting OMP recognition, binding and release. The chaperone SurA is involved in outer membrane protein (OMP) biogenesis in Gram-negative bacteria, but its mechanism of action is not fully understood. Combining mass spectrometric, biophysical and computational approaches, the authors here show how the conformational dynamics of SurA facilitate OMP binding. [ABSTRACT FROM AUTHOR]

Human cytomegalovirus (HCMV) encodes the viral mRNA export factor pUL69, which facilitates the cytoplasmic accumulation of mRNA via interaction with the cellular RNA helicase UAP56 or URH49. We reported previously that pUL69 is phosphorylated by cellular CDKs and the viral CDK-like kinase pUL97. Here, we set out to identify phosphorylation sites within pUL69 and to characterize their importance. Mass spectrometry-based phosphosite mapping of pUL69 identified 10 serine/threonine residues as phosphoacceptors. Surprisingly, only a few of these sites localized to the N terminus of pUL69, which could be due to the presence of additional posttranslational modifications, like arginine methylation. As an alternative approach, pUL69 mutants with substitutions of putative phosphosites were analyzed by Phostag SDS-PAGE. This demonstrated that serines S46 and S49 serve as targets for phosphorylation by pUL97. Furthermore, we provide evidence that phosphorylation of these serines mediates cis/trans isomerization by the prolyl isomerase Pin1, thus forming a functional Pin1 binding motif. Surprisingly, while abrogation of the Pin1 motif did not affect the replication of recombinant cytomegaloviruses, mutation of serines next to the interaction site for UAP56/URH49 strongly decreased viral replication. This was correlated with a loss of UAP56/URH49 recruitment. Intriguingly, the critical serines S13 and S15 were located within a sequence resembling the UAP56 binding motif (UBM) of cellular mRNA adaptor proteins like REF and UIF. We propose that betaherpesviral mRNA export factors have evolved an extended UAP56/URH49 recognition sequence harboring phosphorylation sites to increase their binding affinities. This may serve as a strategy to successfully compete with cellular mRNA adaptor proteins for binding to UAP56/URH49. [ABSTRACT FROM AUTHOR]

Carotid body (CB) type I cells sense the blood PO2 and generate a nervous signal for stimulating ventilation and circulation when blood oxygen levels decline. Three oxygen-sensing enzyme complexes may be used for this purpose: 1) mitochondrial electron transport chain metabolism, 2) heme oxygenase 2 (HO-2)-generating CO, and/or 3) an NAD(P)H oxidase (NOX). We hypothesize that intracellular redox changes are the link between the sensor and nervous signals. To test this hypothesis type I cell autofluorescence of flavoproteins (Fp) and NAD(P)H within the mouse CB ex vivo was recorded as Fp/(Fp-NAD(P)H) redox ratio. CB type I cell redox ratio transiently declined with the onset of hypoxia. Upon reoxygenation, CB type I cells showed a significantly increased redox ratio. As a control organ, the non-oxygen-sensing sympathetic superior cervical ganglion (SCG) showed a continuously reduced redox ratio upon hypoxia. CN, diphenyleneiodonium, or reactive oxygen species influenced chemoreceptor discharge (CND) with subsequent loss of O2 sensitivity and inhibited hypoxic Fp reduction only in the CB but not in SCG Fp, indicating a specific role of Fp in the oxygen-sensing process. Hypoxia-induced changes in CB type I cell redox ratio affected peptidyl prolyl isomerase Pin1, which is believed to colocalize with the NADPH oxidase subunit p47phox in the cell membrane to trigger the opening of potassium channels. We postulate that hypoxia-induced changes in the Fp-mediated redox ratio of the CB regulate the Pin1/p47phox tandem to alter type I cell potassium channels and therewith CND. [ABSTRACT FROM AUTHOR]