Peptidyl-prolyl Isomerase Research Articles

Serine 106 preserves the tertiary structure, function, and stability of a cyclophilin from Staphylococcus aureus

SaCyp, a staphylococcal cyclophilin involved in both protein folding and pathogenesis, has a Ser residue at position 106 and a Trp residue at position 136. While Ser 106 of SaCyp aligned with a cyclosporin A (CsA) binding Ala residue, its Trp 136 aligned with a Trp or a Phe residue of most other cyclophilins. To demonstrate the exact roles of Ser 106 and Trp 136 in SaCyp, we have elaborately studied rCyp[S106A] and rCyp[W136A], two-point mutants of a recombinant SaCyp (rCyp) harboring an Ala substitution at positions 106 and 136, respectively. Of the mutants, rCyp[W136A] showed the rCyp-like CsA binding affinity and peptidyl-prolyl cis-trans isomerase (PPIase) activity. Conversely, the PPIase activity, CsA binding affinity, stability, tertiary structure, surface hydrophobicity, and Trp accessibility of rCyp[S106A] notably differed from those of rCyp. The computational experiments also reveal that the structure, dimension, and fluctuation of SaCyp are not identical to those of SaCyp[S106A]. Furthermore, Ser at position 106 of SaCyp, compared to Ala at the same position, formed a higher number of non-covalent bonds with CsA. Collectively, Ser 106 is an indispensable residue for SaCyp that keeps its tertiary structure, function, and stability intact. Communicated by Ramaswamy H. Sarma

Characterization of Cyclophilin from Thaumarchaeota Nitrosopumilus maritimus: Implications on the Diversity of Chaperone-like Activity in the Archaeal Domain.

The Archaea constitute separate domain of life and show resemblance with bacteria in their metabolic pathways while showing similarity with eukaryotes at the level of molecular processes such as cell division, DNA replication, protein synthesis, and proteostasis. However, the molecular machinery of archaea can be considered a simpler version of that found in eukaryotes because of the absence of multiple paralogs for any given molecular factor. Therefore, archaeal systems can possibly be used as a model system for understanding the eukaryotic protein folding machinery and thereby may help to address the molecular mechanism of various protein (mis)foldings and diseases. In the process of protein folding, the cis–trans isomerization of the peptide–prolyl bond is a rate-limiting step for the correct folding of proteins. Different types of peptidyl–prolyl cis–trans isomerases can accelerate this reaction, e.g., cyclophilin, FKBP, and parvulin. Among the five phyla of the archaeal domain, homologs of the cyclophilin protein are found only in two. Here we have characterized a cyclophilin from an archaeal organism, Nitrosopumilus maritimus (NmCyp), belonging to the phylum Thaumarchaeota. Like other known cyclophilins, NmCyp also possesses PPIase activity that can be inhibited by cyclosporine A. Generally, archaeal proteins are expected to possess differential thermal stability due to their adaptation to extreme environmental niche conditions. However, NmCyp exhibits low thermal stability and starts to aggregate beyond 40 °C. The properties of NmCyp are compared to those reported for the cyclophilin from another archaeal organism, Methanobrevibacter ruminantium. The current study sheds light on the differential behavior of cyclophilin proteins from two different phyla of archaea.

Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 directly binds and stabilizes Nrf2 in breast cancer.

Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1) has been frequently overexpressed in many types of malignancy, suggesting its oncogenic function. It recognizes phosphorylated serine or threonine (pSer/Thr) of a target protein and isomerizes the adjacent proline (Pro) residue, thereby altering folding, subcellular localization, stability, and function of target proteins. The oncogenic transcription factor, Nrf2harbors the pSer/Thr-Pro motif. This prompted us to investigate whether Pin1 could bind to Nrf2 and influence its stability and function in the context of implications for breast cancer development and progression. The correlation between Pin1 and Nrf2 in the triple-negative breast cancer cells was validated by RNASeq analysis as well as immunofluorescence staining. Interaction between Pin1 and Nrf2 was assessed by co-immunoprecipitation and an in situ proximity ligation assay. We found that mRNA and protein levels of Pin1 were highly increased in the tumor tissues of triple-negative breast cancer patients and the human breast cancer cell line. Genetic or pharmacologic inhibition of Pin1 enhanced the ubiquitination and degradation of Nrf2. In contrast, the overexpression of Pin1 resulted in the accumulation of Nrf2 in the nucleus, without affecting its transcription. Notably, the phosphorylation of Nrf2 at serine 215, 408, and 577 is essential for its interaction with Pin1. We also identified phosphorylated Ser104 and Thr277 residues in Keap1, a negative regulator of Nrf2, for Pin1 binding. Pin1 plays a role in breast cancer progression through stabilization and constitutive activation of Nrf2 by competing with Keap1 for Nrf2 binding.

A Redox-Sensitive Cysteine Is Required for PIN1At Function.

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

Determination of Time of Death Based on Blood Ring Formation and RNA Degradation

Time of death (TOD) determination is crucial in criminal cases. The method used to determine the TOD so far is only based on the state of the corpse found, therefore a new method is needed to improve the accuracy of the TOD determination. This study aims to determine the TOD based on a new method, namely the formation of blood rings and RNA degradation. Blood is commonly found in crime scenes. Blood consists of liquid part that is plasma and cellular part consisting of erythrocytes, leukocyte, and thrombocytes. The composition of blood as a liquid that contains dissolved solids makes the drops of dried blood forming “coffee ring effect”, which is a ring-like form on the perimeter of a blood drop. Coffee ring effect is used as an indicator of time by looking at the thickness of the ring formed from the perimeter of the blood drop to the middle which increases with time. RNA degradation was observed using Peptidylprolyl isomerase A (PPIA) gene. The PPIA gene is found in leukocyte and is used to see the degradation of RNA per 30 minutes period using the RT-PCR and qPCR methods. Degradation was observed by comparing the cycle threshold (ct) value of the standard curve with the ct value of the samples per unit time. TOD could be determined by the percentage of the blood ring thickness up until 120th minutes, and by observing the degradation of RNA until the 60th minute, after that the RNA had completely degraded.

Giardia duodenalis and Its Secreted PPIB Trigger Inflammasome Activation and Pyroptosis in Macrophages through TLR4-Induced ROS Signaling and A20-Mediated NLRP3 Deubiquitination.

The extracellular protozoan parasite Giardia duodenalis is a well-known and important causative agent of diarrhea on a global scale. Macrophage pyroptosis has been recognized as an important innate immune effector mechanism against intracellular pathogens. Yet, the effects of noninvasive Giardia infection on macrophage pyroptosis and the associated molecular triggers and regulators remain poorly defined. Here we initially observed that NLRP3 inflammasome-mediated pyroptosis was activated in Giardia-treated macrophages, and inhibition of ROS, NLRP3, or caspase-1 could block GSDMD cleavage, IL-1β, IL-18 and LDH release, and the cell viability reduction. We also confirmed that Giardia-induced NLRP3 inflammasome activation was involved in its K63 deubiquitination. Thus, six candidate deubiquitinases were screened, among which A20 was identified as an effective regulator. We then screened TLRs on macrophage membranes and found that upon stimulation TLR4 was tightly correlated to ROS enhancement, A20-mediated NLRP3 deubiquitination, and pyroptotic signaling. In addition, several Giardia-secreted proteins were predicted as trigger factors via secretome analysis, of which peptidyl-prolyl cis-trans isomerase B (PPIB) independently induced macrophage pyroptosis. This was similar to the findings from the trophozoite treatment, and also led to the TLR4-mediated activation of NLRP3 through K63 deubiquitination by A20. Collectively, the results of this study have significant implications for expanding our understanding of host defense mechanisms after infection with G. duodenalis.

Relationship between brain tissue loss and levels of blood biomarkers in patients with Alzheimer’s disease

AbstractBackgroundThe heme oxygenase‐1 (HO‐1) is known as up‐regulator in AD. The peptidylprolyl isomerase A (PPIA) (also known as cyclophilin A, CyPA) provides protection of neurons against copper‐mediated oxidative stress. The inositol‐requiring enzyme 1 (IRE1) is a major stress transducer in endoplasmic reticulum (ER) stress and abnormal protein aggregation. There is no study to evaluate the relationship between gray matter volume (GMV) loss and the three blood‐based biomarkers. Therefore, in this study, we evaluated the relationship between imaging biomarker by GMV changes and blood biomarkers by plasma levels of HO‐1, PPIA, and IRE1 in the groups of cognitively normal (CN), amnestic mild cognitive impairment (MCI), and AD participants.MethodWe obtained MRI and plasma levels of the three blood biomarkers from 45 CN, 34 amnestic MCI, and 39 AD. To assess the relationship between GMV or WMV loss and the three blood biomarkers, the voxel‐based multiple regression analysis was performed for using all participant data. In addition, region‐of‐interest (ROI)‐based analysis was performed to determine correlations between ROI‐based brain tissue volumes and plasma biomarkers. To investigate the relationship between GMV or WMV at each ROI and the plasma levels of HO‐1, PPIA, and IRE1, we performed the Pearson correlation analyses.ResultThe CypA value was significantly positively correlated with MMSE scores. The HO‐1 value was significantly negatively correlated with age, but was positively correlated with the K‐MMSE score. The IRE1 value was significantly positively correlated with age, but was negatively correlated with K‐MMSE scores. GMV was positively correlated with CypA and HO‐1, but negatively correlated with IRE1.ConclusionOur study demonstrates that subjects with AD have lower circulating levels of HO‐1 than subjects with MCI and the plasma HO‐1 levels were positively associated with the global GMV. This also shows that subjects with AD have lower circulating levels of CyPA than cognitively normal control and the plasma CyPA levels were positively associated with the GMV whereas subjects with AD have higher circulating levels of IRE1 than subjects with MCI and normal control.

Thermococcus sp. KS-1 PPIase as a fusion partner improving soluble production of aromatic amino acid decarboxylase

Peptidyl-prolyl cis-trans isomerase (PPIase, EC 5.2.1.8) catalyzes the racemization reaction of proline residues on a polypeptide chain. This enzyme is also known to function as a molecular chaperon to stabilize protein conformation during the folding process. In this study, we noted FK506 binding protein (FKBP)-type PPIase from a hyperthemophilic archaeon Thermococcus sp. strain KS-1 (PPIase KS−1) to improve the solubility of Pseudomonas putida aromatic amino acid decarboxylase (AADC) that is an indispensable enzyme for fermentative production of plant isoquinoline alkaloids. AADC fused N-terminally with the PPIase KS−1 (PPIase KS−1-AADC), which was synthesized utilizing Escherichia coli host, showed improved solubility and, consequently, the cell-free extract from the recombinant strain exhibited 2.6- to 3.4-fold elevated AADC activity than that from the control strain that expressed the AADC gene without PPIase KS−1. On the other hand, its thermostability was slightly decreased by fusing PPIase KS−1. The recombinant E. coli cells expressing the PPIase KS−1-AADC gene produced dopamine and phenylethylamine from L-dopa and phenylalanine by two- and threefold faster, respectively, as compared with the control strain. We further demonstrated that the efficacy of PPIase KS−1-AADC in solubility and activity enhancement was a little but obviously higher than that of AADC fused N-terminally with NusA protein, which has been assumed to be the most effective protein solubilizer. These results suggest that PPIase KS−1 can be used as one of the best choices for producing heterologous proteins as active forms in E. coli.

Structural basis for recognition of the Hsp90 closed, ATP state by the TPR‐containing co‐chaperone FKBP51

The Hsp90 molecular chaperone drives activation of hundreds of client proteins in the cell through an ATP-dependent conformational cycle guided by distinct co-chaperone regulators. The FKBP51 immunophilin co-chaperone binds Hsp90 through its tetratricopeptide repeat (TPR) domain and catalyzes peptidyl-prolyl isomerase (PPIase) activity during the folding of kinases, nuclear receptors and tau. However, for many TPR cochaperones, the protein:protein interactions and mechanisms which enable Hsp90 specificity and regulation in the folding pathway remain unclear. High resolution structure of the Hsp90:FKBP51:p23 complex was determined using cryo-electron microscopy (EM). Native gel electrophoresis, gel filtration chromatography coupled with multiangle light scattering, and negative stain-EM were used to test interaction of FKBP51/p23 with Hsp90 in different nucleotide binding states. The structure of the Hsp90:FKBP51:p23 complex was verified with mutational analysis and site-specific photocrosslinking experiments. We developed in vitro conditions that promote and stabilize the closed, ATP conformation of Hsp90, enabling characterization of binding interactions between defined open (apo) and closed (ATP) states. We find that FKBP51 preferentially binds the closed, ATP state, forming a complex with a 2:1 stoichiometry that is further stabilized by p23 binding. Using cryo-EM we determined a 3.3 Å structure the Hsp90:FKBP51:p23 complex that reveals distinct contacts by the TPR domain of FKBP51 that define its asymmetric interaction and recognition of C-terminal Hsp90 dimer. We propose a model in which helix 7 of the TPR domain of FKBP51 functions as a critical specificity element for engaging Hsp90 in the p23-stabilized, ATP state to promote client maturation. This arrangement flexibly positions the FK1 domain of FKBP51 adjacent Hsp90 client binding sites in a manner we predict facilitates site-specific PPIase activity towards clients in coordination with the Hsp90 chaperone cycle.

Pin1 as Molecular Switch in Vascular Endothelium: Notes on Its Putative Role in Age-Associated Vascular Diseases.

By controlling the change of the backbones of several cellular substrates, the peptidyl-prolyl cis-trans isomerase Pin1 acts as key fine-tuner and amplifier of multiple signaling pathways, thereby inducing several biological consequences, both in physiological and pathological conditions. Data from the literature indicate a prominent role of Pin1 in the regulating of vascular homeostasis. In this review, we will critically dissect Pin1’s role as conformational switch regulating the homeostasis of vascular endothelium, by specifically modulating nitric oxide (NO) bioavailability. In this regard, Pin1 has been reported to directly control NO production by interacting with bovine endothelial nitric oxide synthase (eNOS) at Ser116-Pro117 (human equivalent is Ser114-Pro115) in a phosphorylation-dependent manner, regulating its catalytic activity, as well as by regulating other intracellular players, such as VEGF and TGF-β, thereby impinging upon NO release. Furthermore, since Pin1 has been found to act as a critical driver of vascular cell proliferation, apoptosis, and inflammation, with implication in many vascular diseases (e.g., diabetes, atherosclerosis, hypertension, and cardiac hypertrophy), evidence indicating that Pin1 may serve a pivotal role in vascular endothelium will be discussed. Understanding the role of Pin1 in vascular homeostasis is crucial in terms of finding a new possible therapeutic player and target in vascular pathologies, including those affecting the elderly (such as small and large vessel diseases and vascular dementia) or those promoting the full expression of neurodegenerative dementing diseases.

Abstract 14033: Inhibition of the Prolyl Isomerase Pin1 Improves Endothelial Function and Attenuates Vascular Remodelling in Pulmonary Hypertension by Inhibiting TGF-β Signalling

Introduction: Pulmonary arterial hypertension (PAH) is a devastating disease, characterized by obstructive pulmonary vascular remodelling ultimately leading to right ventricular (RV) failure and death. Disturbed transforming growth factor-β (TGF-β) / bone morphogenetic protein (BMP) signalling, endothelial cell dysfunction, increased proliferation of smooth muscle cells and fibroblasts, and inflammation contribute to this abnormal remodelling. Peptidyl prolyl isomerase Pin1 has been identified as a critical driver of proliferation and inflammation in vascular cells, but its role in the disturbed TGF-β/BMP signalling, endothelial cell dysfunction and vascular remodelling in PAH is unknown. Methods and Results: Pin1 expression is increased in cultured pulmonary microvascular endothelial cells (MVECs) and lung tissue of PAH patients. Pin1 inhibitor, juglone significantly decreased TGF-β signalling, increased BMP signalling, normalized their hyper-proliferative, and inflammatory phenotype. Juglone treatment reversed vascular remodelling through reducing TGF-β signalling in monocrotaline + shunt-PAH rat model. Juglone treatment decreased Fulton index, but did not affect or harm cardiac function and remodelling in rats with RV pressure load induced by pulmonary artery banding. Conclusions: Our study demonstrates that inhibition of Pin1 reversed the PAH phenotype in PAH-MVECs in vitro and in PAH rats in vivo, potentially through modulation of TGF-β/BMP signalling pathways. Selective inhibition of Pin1 could be a novel therapeutic option for the treatment of PAH.

Genetic and Proteomic Basis of Sclerotinia Stem Rot Resistance in Indian Mustard [Brassica juncea (L.) Czern & Coss.

Sclerotinia stem rot is one of the utmost important disease of mustard, causing considerable losses in seed yield and oil quality. The study of the genetic and proteomic basis of resistance to this disease is imperative for its effective utilization in developing resistant cultivars. Therefore, the genetic pattern of Sclerotinia stem rot resistance in Indian mustard was studied using six generations (P1, P2, F1, F2, BC1P1, and BC1P2) developed from the crossing of one resistant (RH 1222-28) and two susceptible (EC 766300 and EC 766123) genotypes. Genetic analysis revealed that resistance was governed by duplicate epistasis. Comparative proteome analysis of resistant and susceptible genotypes indicated that peptidyl-prolyl cis-trans isomerase (A0A078IDN6 PPIase) showed high expression in resistant genotype at the early infection stage while its expression was delayed in susceptible genotypes. This study provides important insight to mustard breeders for designing effective breeding programs to develop resistant cultivars against this devastating disease.

Adsorptive Microtiter Plates As Solid Supports in Affinity Purification Workflows.

Affinity ligands such as antibodies are widely used in (bio)medical research for purifying proteins from complex biological samples. These ligands are generally immobilized onto solid supports which facilitate the separation of a captured protein from the sample matrix. Adsorptive microtiter plates are commonly used as solid supports prior to immunochemical detection (e.g., immunoassays) but hardly ever prior to liquid chromatography–mass spectrometry (LC-MS-)-based detection. Here, we describe the use of adsorptive microtiter plates for protein enrichment prior to LC-MS detection, and we discuss opportunities and challenges of corresponding workflows, based on examples of targeted (i.e., soluble receptor for advanced glycation end-products (sRAGE) in human serum) and discovery-based workflows (i.e., transcription factor p65 (NF-κB) in lysed murine RAW 264.7 macrophages and peptidyl-prolyl cis–trans isomerase FKBP5 (FKBP5) in lysed human A549 alveolar basal epithelial cells). Thereby, we aim to highlight the potential usefulness of adsorptive microtiter plates in affinity purification workflows prior to LC-MS detection, which could increase their usage in mass spectrometry-based protein research.

CD16 and CD56 mRNA Expression in Decidua of Patients with Missed and Spontaneous Abortions

Study Objective to estimate relative mRNA expression of receptors CD16 and CD56 in decidual tissue of the patients with missed and spontaneous abortions. Design Prospective cohort study. Setting Academic affiliated tertiary care center. Patients or Participants Patients with spontaneous abortions (n=22), missed abortions (n=22) and women with normal pregnancy, admitted for artificial abortions (control group, n=57) were examined at 6-10 weeks of gestation. Patients with severe extragenital diseases, antiphospholipid syndrome, endocrine disorders, uterine malformations, and fetal chromosomal abnormalities were excluded from the research. Interventions Uterine abrasion. Measurements and Main Results Decidual tissue was obtained by uterine abrasion. Relative mRNA expression of CD16 and CD56 was defined by reverse transcription-quantitative polymerase chain reaction (RT-PCR). Peptidylprolyl isomerase А and beta-actin were used as housekeeping genes. Expression of mRNA was counted in relative units by ΔΔCq method. Data were performed as the median and interquartile range and compared using Kruskal-Wallis and Mann-Whitney tests (GraphPad Prism 9.0.2). Relative mRNA expression of CD56 (a marker of natural killer (NK) cells) in decidual tissue of the patients with spontaneous and missed abortions didn't have significant differences with the control group, which confirms the data of meta-analysis (Seshadri S., Sunkara S.K., 2013). Relative mRNA expression of CD16 in decidua of patients with missed abortions (837,53 (313,00; 1082,39)) was lower compared to women with spontaneous abortions (1077 (510,23; 1541,37)) and the control group (1692,57 (1243,34; 2759,13)) (p<0,0001 and p<0,00001, respectively). Expression of CD16 in decidua of the patients with spontaneous abortions was lower, than in the control group (p<0,001). CD16 (FcγRIII) is involved in antibody-dependent cellular cytotoxicity. Uterine NK cells have CD56 + CD16- phenotype (Tang A.W., 2011). The main cells in the decidua expressing CD16 are monocytes, macrophages (Ning F. et al., 2016), and neutrophils (Presicce P. et al., 2018). Conclusion CD16 can play an important role in the pathogenesis of early miscarriages. to estimate relative mRNA expression of receptors CD16 and CD56 in decidual tissue of the patients with missed and spontaneous abortions. Prospective cohort study. Academic affiliated tertiary care center. Patients with spontaneous abortions (n=22), missed abortions (n=22) and women with normal pregnancy, admitted for artificial abortions (control group, n=57) were examined at 6-10 weeks of gestation. Patients with severe extragenital diseases, antiphospholipid syndrome, endocrine disorders, uterine malformations, and fetal chromosomal abnormalities were excluded from the research. Uterine abrasion. Decidual tissue was obtained by uterine abrasion. Relative mRNA expression of CD16 and CD56 was defined by reverse transcription-quantitative polymerase chain reaction (RT-PCR). Peptidylprolyl isomerase А and beta-actin were used as housekeeping genes. Expression of mRNA was counted in relative units by ΔΔCq method. Data were performed as the median and interquartile range and compared using Kruskal-Wallis and Mann-Whitney tests (GraphPad Prism 9.0.2). Relative mRNA expression of CD56 (a marker of natural killer (NK) cells) in decidual tissue of the patients with spontaneous and missed abortions didn't have significant differences with the control group, which confirms the data of meta-analysis (Seshadri S., Sunkara S.K., 2013). Relative mRNA expression of CD16 in decidua of patients with missed abortions (837,53 (313,00; 1082,39)) was lower compared to women with spontaneous abortions (1077 (510,23; 1541,37)) and the control group (1692,57 (1243,34; 2759,13)) (p<0,0001 and p<0,00001, respectively). Expression of CD16 in decidua of the patients with spontaneous abortions was lower, than in the control group (p<0,001). CD16 (FcγRIII) is involved in antibody-dependent cellular cytotoxicity. Uterine NK cells have CD56 + CD16- phenotype (Tang A.W., 2011). The main cells in the decidua expressing CD16 are monocytes, macrophages (Ning F. et al., 2016), and neutrophils (Presicce P. et al., 2018). CD16 can play an important role in the pathogenesis of early miscarriages.

Qualitative and quantitative proteomic approach of epicardial cell secretome from atrial fibrillation patients after cholinergic stimulation

Abstract Background The association between epicardial adipose tissue (EAT) and atrial fibrillation (AF) was already described by several authors. In fact, botulinum toxin injection in this tissue reduces new-onset AF after open-heart surgery. Our previous results showed an elevation of adiposity in epicardial stromal vascular cells (SVC), assessed by the higher levels of fatty acid-binding protein 4 (FABP4) in AF patients; and a higher lipid accumulation in these cells after chronic acetylcholine treatment. We wanted to identify the proteins released from EAT SVC in presence/absence of mature adipocytes, that could be exerting a paracrine effect over the myocardium. The identification of these proteins might shed light on possible triggers in epicardial SVC and the mechanisms underlying botulinum toxin benefits on AF. Material and methods Proteomic studies were performed in 32 samples from 8 patients undergoing open-heart surgery (4 with and 4 without AF). Epicardial SVC were isolated by collagenase digestion and cultured in M199 medium. Then, cells were induced or not to adipocyte differentiation. After this intervention, cells were treated or not with acetylcholine (10uM) for 30 min. Conditioned medium was stored until be used. Differential released proteins were identified by nano-high performance liquid chromatography (HPLC) and Triple-Time of flying (TOF) analysis, and quantified by SWATH-Ms proteomics anaylisis. Results The quantitative proteomic approach has identified 111 common proteins in EAT SVC from patients with and without AF. A 79,3% of the genes which encoded the proteins identified were citoplasmic. A 78,4% were classified as components of the cellular exosomes, followed by genes related with centrosome (37,1%), nucleosome (15,5%), lysosomes (40,5%) and nucleolus (37,1%). Acute cholinergic treatment with ACh at 10 uM decreased α-defensin 3 (DEFA3, ID: 59666; p-value = 0,0297) secretion from EAT SVC of AF patients in comparison with EAT SVC from non-AF patients. In the same line, Peptidyl-prolyl cis-trans isomerase A (PPIA, ID: P62937) showed a lower secretion from SVC of AF patients (p=0,0326). After adipogenesis-induction, adipocyte presence modified the protein secretion under ACh treatment: Differences between AF and non-AF patients lied on 2 different proteins: profilin 1 (PFN1, ID: P07737, p-value = 0,0286) and β-enolase (ENO3, ID: P13929, p-value = 0,0414), showing a higher and lower secretion in AF patients regarding non-AF patients, respectively. Conclusions EAT SVC showed a differential protein secretion according adipocyte and AF presence. Although further studies are needed, the proteins differentially secreted in EAT SVC are related to inflammation (DEFA3), structure (PFN1) and glucose metabolism (ENO3), pointing the pathways that could be modified in AF patients. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Plan Estatal de I+D+I 2016-2020 and ISCIII-Subdirecciόn General de Evaluaciόn y Fomento de la Investigaciόn el Fondo Europeo de Desarrollo Regional (FEDER) Figure 1. Common proteins secreted from EAT SVCFigure 2. Differentially secreted proteins in AF

Distribution of Peptidyl-Prolyl Isomerase (PPIase) in the Archaea.

Cis-trans isomerization of the peptide bond prior to proline is an intrinsically slow process but plays an essential role in protein folding. In vivo cis-trans isomerization reaction is catalyzed by Peptidyl-prolyl isomerase (PPIases), a category of proteins widely distributed among all the three domains of life. The present study is majorly focused on the distribution of different types of PPIases in the archaeal domain. All the three hitherto known families of PPIases (namely FKBP, Cyclophilin and parvulin) were studied to identify the evolutionary conservation across the phylum archaea. The basic function of cyclophilin, FKBP and parvulin has been conserved whereas the sequence alignment suggested variations in each clade. The conserved residues within the predicted motif of each family are unique. The available protein structures of different PPIase across various domains were aligned to ascertain the structural variation in the catalytic site. The structural alignment of native PPIase proteins among various groups suggested that the apo-protein may have variable conformations but when bound to their specific inhibitors, they attain similar active site configuration. This is the first study of its kind which explores the distribution of archaeal PPIases, along with detailed structural and functional analysis of each type of PPIase found in archaea.

Serum Peptidyl-prolyl Cis-trans Isomerase NIMA-interacted 1 (Pin1) as a Non-invasive Marker for Liver Fibrosis due to Chronic Hepatitis C Virus.

Background: Hepatitis C virus (HCV) may remain asymptomatic or cause liver fibrosis and cirrhosis. Objectives: We aimed to assess the relationship between serum peptidyl-prolyl cis-trans isomerase NIMA-interacted 1 (Pin1) levels and liver fibrosis due to HCV. Methods: Serum samples of successive patients with HCV genotype 1b and healthy volunteers were collected, and Pin1 levels were measured using ELISA kits. Liver fibrosis stages were calculated by the Ishak Scoring System and subdivided into two groups; stage &lt; 3 (mild fibrosis) and ≥ 3 (advanced fibrosis). Correlation and area under receiver operating characteristics (AUROC) analysis were used to investigate the relationship between Pin1 and clinical and histopathological properties of HCV infection. Results: Ninety-four patients with HCV and 47 age- and sex-matched volunteers were included. The median age of the participants was 52, and 55% of whom were females. The mean (SD) of Pin1 serum level was significantly higher in the HCV group compared with healthy volunteers (33.94 (21.15) vs. 26.82 (8.85) pg/mL, respectively, P = 0.007). Seventy-seven (82%) and 17 (18%) of the participants showed mild and advanced fibrosis, respectively. Pin1 serum levels were significantly lower in the mild compared with advanced fibrosis group (29 (17.88) vs. 43.59 (7.98) pg/mL, respectively, P &lt; 0.001). We found a significantly positive correlation between Pin1 serum level and liver fibrosis stage (r = 0.71, P &lt; 0.001). The cut off of 33.04 pg/mL of Pin1 serum level showed the best sensitivity (100%) and specificity (68.4%) (AUROC = 0.81 [95% confidence interval: 0.72 - 0.90], P &lt; 0.001) for distinguishing advanced from mild liver fibrosis. Conclusions: Serum Pin1 level may be a relevant marker for predicting liver fibrosis in HCV infected patients.

Unique virulence role of post-translocational chaperone PrsA in shaping Streptococcus pyogenes secretome

ABSTRACT Streptococcus pyogenes (group A Streptococcus, GAS) is a strict human pathogen causing a broad spectrum of diseases and a variety of autoimmune sequelae. The pathogenesis of GAS infection mostly relies on the production of an extensive network of cell wall-associated and secreted virulence proteins, such as adhesins, toxins, and exoenzymes. PrsA, the only extracellular parvulin-type peptidyl-prolyl isomerase expressed ubiquitously in Gram-positive bacteria, has been suggested to assist the folding and maturation of newly exported proteins to acquire their native conformation and activity. Two PrsA proteins, PrsA1 and PrsA2, have been identified in GAS, but the respective contribution of each PrsA in GAS pathogenesis remains largely unknown. By combining comparative proteomic and phenotypic analysis approaches, we demonstrate that both PrsA isoforms are required to maintain GAS proteome homeostasis and virulence-associated traits in a unique and overlapping manner. The inactivation of both PrsA in GAS caused remarkable impairment in biofilm formation, host adherence, infection-induced cytotoxicity, and in vivo virulence in a murine soft tissue infection model. The concordance of proteomic and phenotypic data clearly features the essential role of PrsA in GAS full virulence.

Role of peptidyl-prolyl &lt;i&gt;cis–trans&lt;/i&gt; isomerases in infectious diseases and host–pathogen interactions

Peptidyl-prolyl cis-trans isomerases (PPIases) catalyse the cis-trans isomerization of Cα atoms about the peptide bond preceding a proline residue, thereby regulating a conformational switch which controls a plethora of cellular processes. PPIases play a key role in the survival, reproduction, proliferation and virulence of microbial pathogens vis-a-vis their human host. In addition, human PPIases either aid or retard viral replication and modulate host immune response. The article discusses the structure-function relationships of PPIases in the context of microbial virulence (with an emphasis on viruses), and on targeting PPIases for COVID-19, responsible for untold human sufferings. [ABSTRACT FROM AUTHOR] Copyright of Current Science (00113891) is the property of Indian Academy of Sciences and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

MiR-23a contributes to T cellular redox metabolism in juvenile idiopathic oligoarthritis.

JIA is a chronic inflammatory disease of unknown origin. The regulation of inflammatory processes involves multiple cellular steps including mRNA transcription and translation. Different miRNAs control these processes tightly. We aimed to determine the roles of specific miRNAs within JIA pathogenesis. We performed a global miRNA expression analysis in parallel in cells from the arthritic joint and peripheral blood of oligoarticular JIA patients and healthy controls. Quantitative RT-PCR analysis was used to verify expression of miRNA in T cells. Ex vivo experiments and flow cytometric analyses were used to analyse proliferation and redox metabolism. Global miRNA expression analysis demonstrated a different composition of miRNA expression at the site of inflammation compared with peripheral blood. Bioinformatic analysis of predicted miRNA target genes suggest a huge overrepresentation of genes involved in metabolic and oxidative stress pathways in the inflamed joint. Despite enhanced reactive oxygen species (ROS) levels within the local inflammatory milieu, JIA T cells are hyperproliferative and reveal an overexpression of miR-23a, which is an inhibitor of Peptidyl-prolyl isomerase F (PPIF), the regulator of mitochondrial ROS escape. Mitochondrial ROS escape is diminished in JIA T cells, resulting in their prolonged survival. Our data suggest that miRNA-dependent mitochondrial ROS shuttling might be a mechanism that contributes to T cell regulation in JIA at the site of inflammation.