Epsilon Protein Research Articles

Relationship between Urinary Alzheimer-Associated Neuronal Thread Protein and Apolipoprotein Epsilon 4 Allele in the Cognitively Normal Population.

We investigated the relationship between urinary Alzheimer-associated neuronal thread protein (AD7c-NTP) levels and apolipoprotein epsilon 4 (ApoE ɛ4) alleles, as well as other factors that cause cognitive decline, in the cognitively normal population. We recruited 329 cognitively normal right-handed Han Chinese subjects who completed ApoE gene testing and urinary AD7c-NTP testing. There was no significant difference in urinary AD7c-NTP levels between the normal control and subjective cognitive decline groups. Urinary AD7c-NTP levels were significantly higher in subjects with ApoE ɛ3/4 and 4/4 [0.6074 (0.6541) ng/mL] than in subjects without ApoE ɛ4 [0.4368 (0.3392) ng/mL and 0.5287 (0.3656) ng/mL], and urinary AD7c-NTP levels positively correlated with ApoE genotype grade (r = 0.165, p = 0.003). There were significant differences in urinary AD7c-NTP levels between subjects with and without a history of coronary heart disease or diabetes. Urinary AD7c-NTP levels were not related to years of education, nature of work, family history of dementia, a history of hypertension, stroke, anemia, or thyroid dysfunction. Urinary AD7c-NTP levels were positively correlated with ApoE grade in the cognitively normal population. The relationship between risk factors of cognitive decline and urinary AD7c-NTP levels provides a new way for us to understand AD and urinary AD7c-NTP.

Brain Abscess as Severe Presentation of Specific Granule Deficiency

Severe invasive infections such as brain abscess in a child should prompt an immune evaluation. Specific granule deficiency (SGD) is a rare morphologic neutrophil granular defect characterized by reduced granules within neutrophils, absence of granule proteins, and bilobed nuclei. Patients are susceptible to invasive bacterial infections and Candida infections. Mutations in CCAT/enhancer binding protein epsilon (C/EBP-ε) are the most commonly described cause of SGD. The dihydrorhodamine assay is a quantitative and qualitative functional test that determines the oxidative burst and killing potential of neutrophils. Herein, we describe two brothers with specific granule deficiency. The index patient had a history of cellulitis twice in the first year of life and then presented at 13 months age with fever, leukocytosis, and right sided weakness. A large space occupying brain abscess was diagnosed. He underwent surgical drainage and cultures yielded Staphylococcus aureus. This infection prompted his diagnosis. His older brother had also been healthy but too had had several episodes of cellulitis. His brother too was diagnosed with SGD when family genetic screening was performed. Evaluation of the index patient included a peripheral smear that showed absent neutrophil granule presence. Forward and side scatter of whole blood via flow cytometry revealed a loss of granularity of neutrophils. A DHR was performed to rule out functional killing defects. After stimulation with PMA, neutrophils from the index patient displayed three distinct patterns, two with abnormal oxidase production, and two with reduced function. Both patients were ultimately diagnosed with SGD and remain on lifelong anti-bacterial prophylaxis. Diagnosis of SGD relies on establishing reduced or absent granularity within neutrophils. Lifelong anti-bacterial and anti-fungal prophylaxis is indicated. Hematopoietic cell transplantation has also been curative.

Ingenuity pathway analysis of human facet joint tissues: Insight into facet joint osteoarthritis.

Facet joint osteoarthritis (FJOA) is a common degenerative joint disorder with high prevalence in the elderly. FJOA causes lower back pain and lower extremity pain, and thus severely impacts the quality of life of affected patients. Emerging studies have focused on the histomorphological and histomorphometric changes in FJOA. However, the dynamic genetic changes in FJOA have remained to be clearly determined. In the present study, previously obtained RNA deep sequencing data were subjected to an ingenuity pathway analysis (IPA) and canonical signaling pathways of differentially expressed genes (DEGs) in FJOA were studied. The top 25 enriched canonical signaling pathways were identified and canonical signaling pathways with high absolute values of z-scores, specifically leukocyte extravasation signaling, Tec kinase signaling and osteoarthritis pathway, were investigated in detail. DEGs were further categorized by disease, biological function and toxicity (tox) function. The genetic networks between DEGs as well as hub genes in these functional networks were also investigated. It was demonstrated that C-X-C motif chemokine ligand 8, elastase, neutrophil expressed, growth factor independent 1 transcriptional repressor, Spi-1 proto-oncogene, CCAAT enhancer binding protein epsilon, GATA binding protein 1, TAL bHLH transcription factor 1, erythroid differentiation factor, minichromosome maintenance complex component 4, BTG anti-proliferation factor 2, BRCA1 DNA repair-associated, cyclin D1, chromatin assembly factor 1 subunit A, triggering receptor expressed on myeloid cells 1 and tumor protein p63 were hub genes in the top 5 IPA networks (with a score >30). The present study provides insight into the pathological processes of FJOA from a genetic perspective and may thus benefit the clinical treatment of FJOA.

Lung endothelial cells are sensitive to epsilon toxin from Clostridium perfringens

The pore-forming protein epsilon toxin (Etx) from Clostridium perfringens produces acute perivascular edema affecting several organs, especially the brain and lungs. Despite the toxin evident effect on microvasculature and endothelial cells, the underlying molecular and cellular mechanisms remain obscure. Moreover, no Etx-sensitive endothelial cell model has been identified to date. Here, we characterize the mouse lung endothelial cell line 1G11 as an Etx-sensitive cell line and compare it with the well-characterized Etx-sensitive Madin-Darby canine kidney epithelial cell line. Several experimental approaches, including morphological and cytotoxic assays, clearly demonstrate that the 1G11 cell line is highly sensitive to Etx and show the specific binding, oligomerization, and pore-forming activity of the toxin in these cells. Recently, the myelin and lymphocyte (MAL) protein has been postulated as a putative receptor for Etx. Here, we show the presence of Mal mRNA in the 1G11 cell line and the presence of the MAL protein in the endothelium of some mouse lung vessels, supporting the hypothesis that this protein is a key element in the Etx intoxication pathway. The existence of an Etx-sensitive cell line of endothelial origin would help shed light on the cellular and molecular mechanisms underlying Etx-induced edema and its consequences.

Remarkable Homeostasis of Protein Sialylation in Skeletal Muscles of Hibernating Daurian Ground Squirrels (Spermophilus dauricus)

As the most common post-translational protein modification, glycosylation is intimately linked to muscle atrophy. This study aimed to investigate the performance of protein glycosylation in the soleus muscle (SOL) in Daurian ground squirrels (Spermophilus dauricus) and to determine the potential role of protein glycosylation in the mechanism underlying disuse muscle atrophy prevention. The results showed that (1) seven glycan structures comprising sialic acid α2-3 galactose (SAα2-3Gal) were altered during hibernation; (2) alterations in the SAα2-3Gal structure during hibernation were based on changes in the expression levels of beta-galactoside alpha-2 and 3-sialyltransferases; and (3) α2-3–linked sialylated modifications of heat shock cognate 70 and pyruvate kinase and expression of 14-3-3 epsilon protein were oscillatorily changed during hibernation. Our findings indicate that the skeletal muscles of hibernating Daurian ground squirrels maintain protein sialylation homeostasis by restoring sialylation modification during periodic interbout arousal, which might protect the skeletal muscles against disuse atrophy.

Progress of human CCAAT/enhancer binding protein epsilon on inflammation and infection

Inflammation and infection are main causes of death in critically ill patients. But traditional treatment are non-comprehensive and limited. Human CCAAT/enhancer binding protein epsilon (C/EBPε) is a key transcription factor regulating the terminal differentiation of neutrophils. It plays an important role in anti-inflammatory and anti-infective process by regulating inflammatory response cells. This article reviews the changes of C/EBPε in inflammation and infection, related regulatory mechanisms and targeted reversal measures, in order to provide references for exploring new directions and effective measures for inflammation and infection treatment.

Functional study of 14-3-3 protein epsilon (YWHAE) in keratinocytes: microarray integrating bioinformatics approaches

Previously, we detected that 14-3-3 protein epsilon (YWHAE) was involved in the pathogenesis of atopic dermatitis (AD) and tyrosinase-mediated pigmentation. In this study, we aimed to identify critical factors associated with YWHAE in human keratinocytes using high-throughput screening (HTS) approaches to reveal its functions in skin. We overexpressed YWHAE in human HaCaT keratinocytes and then conducted serial HTS studies, including RNA sequencing integrated with antibody arrays and the implementation of bioinformatics algorithms. Cumulatively, these approaches identified several novel genes in keratinocytes associated with the function of YWHAE including KRT9, KRT1, KRT6C, BST2, CIB2, APH1B, ACTC1, IFI27, TUBA1A, CAPN6, UTY, MX2, and MAPK15, based on RNA sequencing data, and MAPK1, MMP2, TYK2, NOS3, and CASP3, based on antibody array data. In particular, CD37 is a unique gene that was detected and validated in all the methods applied in this study. By integrating the datasets obtained from these HTS studies and utilizing the strengths of each method, we obtained new insights into the functional role of YWHAE in skin keratinocytes. The approach used here could contribute to the clinical understanding of YWHAE-associated applications in the treatment of AD disease.AbbreviationsDAVIDthe database for annotation, visualization and integrated discoveryHTSHigh-throughput screeningKEGGKyoto Encyclopedia of Genes and GenomesPPIprotein-protein interactionsCommunicated by Ramaswamy H. Sarma

Development of multi-epitope driven subunit vaccine against Fasciola gigantica using immunoinformatics approach

Fascioliasis, a serious helminth disease of the livestock population, results from infection with the parasite Fasciola. Despite the alarming increase in drug resistance, a safe and fully effective vaccine for fascioliasis is still not available. In the present study, we employed high-throughput immunoinformatics approaches to design a multi-epitope based subunit vaccine using seven important F. gigantica proteins (cathepsin B, cathepsin L, leucyl aminopeptidase, thioredoxin glutathione reductase, fatty acid binding protein-1, saposin-like protein-2, and 14–3-3 protein epsilon). The CTL, HTL, and B-cell epitopes were selected for designing the vaccine on the basis of their immunogenic behavior and binding affinity. The engineered vaccine showed potential immunogenic efficacy by elaborating the IFN-γ and humoral response. The modeled structure of the vaccine was docked with the toll-like receptor-2 immune receptor, and the molecular dynamics simulation was performed to understand the stability, interaction, and dynamics of the complex. Finally, in silico cloning of the resulting vaccine was performed to create the plasmid construct of vaccine for expression in an appropriate biological system. Experimental evaluation of the designed vaccine construct in an animal model may result in a novel and immunogenic vaccine that may confer protection against F. gigantica infection.

Aldosterone rapidly activates p-PKC delta and GPR30 but suppresses p-PKC epsilon protein levels in rat kidney.

Aldosterone rapidly enhances protein kinase C (PKC) alpha and beta1 proteins in the rat kidney. The G protein-coupled receptor 30 (GPR30)-mediated PKC pathway is involved in the inhibition of the potassium channel in HEK-239 cells. GPR30 mediates rapid actions of aldosterone in vitro. There are no reports available regarding the aldosterone action on other PKC isoforms and GPR30 proteins in vivo. The aim of the present study was to examine rapid actions of aldosterone on protein levels of phosphorylated PKC (p-PKC) delta, p-PKC epsilon, and GPR30 simultaneously in the rat kidney. Male Wistar rats were intraperitoneally injected with normal saline solution or aldosterone (150 µg/kg body weight). After 30 minutes, abundance and immunoreactivity of p-PKC delta, p-PKC epsilon, and GPR30 were determined by Western blot analysis and immunohisto-chemistry, respectively. Aldosterone administration significantly increased the renal protein abundance of p-PKC delta by 80% (p<0.01) and decreased p-PKC epsilon protein by 50% (p<0.05). Aldosterone injection enhanced protein immunoreactivity of p-PKC delta but suppressed p-PKC epsilon protein intensity in both kidney cortex and medulla. Protein abundance of GPR30 was elevated by aldosterone treatment (p<0.05), whereas the immunoreactivity was obviously changed in the kidney cortex and inner medulla. Aldosterone translocated p-PKC delta and GPR30 proteins to the brush border membrane of proximal convoluted tubules. This is the first in vivo study simultaneously demonstrating that aldosterone administration rapidly elevates protein abundance of p-PKC delta and GPR30, while p-PKC epsilon protein is suppressed in rat kidney. The stimulation of p-PKC delta protein levels by aldosterone may be involved in the activation of GPR30.

C/EBPε ΔRS derived from a neutrophil-specific granule deficiency patient interacts with HDAC1 and its dysfunction is restored by trichostatin A

CCAAT/enhancer binding protein epsilon (C/EBPε), a myeloid-specific transcription factor, plays an important role in granulopoiesis. A loss-of-function mutation in this protein can result in an abnormal development of neutrophils and eosinophils, known as neutrophil-specific granule deficiency (SGD). The transcriptional activity of C/EBPε is regulated by interactions with other transcription factors and/or post-translational modification, including acetylation. Previously, we reported a novel SGD patient who had a homozygous mutation for two amino acids, arginine (R247) and serine (S248), which were deleted in the basic leucine zipper domain of C/EBPε (ΔRS) and exhibited loss of transcriptional activity with aberrant protein–protein interactions. In the present study, we found that a single amino acid deletion of either R247 (ΔR) or S248 (ΔS) was sufficient for the loss of C/EBPε transcriptional activity, while an amino acid substitution at S248 to alanine in C/EBPε (SA) had comparable transcriptional activity with the wild-type C/EBPε (WT). Although acetylation at lysine residues (K121 and K198) is indispensable for C/EBPε transcriptional activity, an acetylation mimic form of ΔRS (ΔRS-K121/198Q) did not exhibit the transcriptional activity. Interestingly, we discovered that ΔRS, ΔR, ΔS, and ΔRS-K121/198Q interacted with histone deacetylase 1 (HDAC1), whereas WT and SA did not. Furthermore, the proteoglycan 2/eosinophil major basic protein induction activity of ΔRS, ΔR, and ΔS could be restored by the HDAC inhibitor, trichostatin A (TSA), and protein–protein interactions between ΔRS and Gata1 could also be recovered by TSA treatment. Taken together, our results show that TSA has the potential to restore the transcriptional activity of ΔRS, indicating that the inhibition of HDAC1 could be a molecularly targeted treatment for SGD with ΔRS.

Differentially expressed mRNAs, proteins and miRNAs associated to energy metabolism in skeletal muscle of beef cattle identified for low and high residual feed intake

BackgroundFeed efficiency is one of the most important parameters that affect beef production costs. The energy metabolism of skeletal muscle greatly contributes to variations in feed efficiency. However, information regarding differences in proteins involved in the energy metabolism of the skeletal muscle in beef cattle divergently identified for feed efficiency is scarce. In this study, we aimed to investigate energy metabolism of skeletal muscle of Nellore beef cattle, identified for low and high residual feed intake using a proteomics approach. We further assessed the expression of candidate microRNAs as a one of the possible mechanisms controlling the biosynthesis of the proteins involved in energy metabolism that were differentially abundant between high and low residual feed intake animals.ResultsA greater abundance of 14–3-3 protein epsilon (P = 0.01) was observed in skeletal muscle of residual feed intake (RFI) high animals (RFI-High). Conversely, a greater abundance of Heat Shock Protein Beta 1 (P < 0.01) was observed in the skeletal muscle of RFI-Low cattle. A greater mRNA expression of YWHAE, which encodes the 14–3-3 protein epsilon, was also observed in the skeletal muscle of RFI-High animals (P = 0.01). A lower mRNA expression of HSPB1, which encodes the Heat Shock Protein Beta 1, was observed in the skeletal muscle of RFI-High animals (P = 0.01). The miR-665 was identified as a potential regulator of the 14–3-3 protein epsilon, and its expression was greater in RFI-Low animals (P < .001). A greater expression of miR-34a (P = 0.01) and miR-2899 (P < .001) was observed in the skeletal muscle of RFI-High animals, as both miRNAs were identified as potential regulators of HSPB1 expression.ConclusionOur results show that Nellore cattle divergently identified for feed efficiency by RFI present changes in the abundance of proteins involved in energy expenditure in skeletal muscle. Moreover, our data point towards that miR-665, miR34a and miR-2899 are likely involved in controlling both 14-3-3 epsilon and HSPB1 proteins identified as differentially abundant in the skeletal muscle of RFI-High and RFI-Low Nellore cattle.

Gain-of-function CEBPE mutation causes noncanonical autoinflammatory inflammasomopathy

CCAAT enhancer-binding protein epsilon (C/EBPε) is a transcription factor involved in late myeloid lineage differentiation and cellular function. The only previously known disorder linked to C/EBPε is autosomal recessive neutrophil-specific granule deficiency leading to severely impaired neutrophil function and early mortality. The aim of this study was to molecularly characterize the effects of C/EBPε transcription factor Arg219His mutation identified in a Finnish family with previously genetically uncharacterized autoinflammatory and immunodeficiency syndrome. Genetic analysis, proteomics, genome-wide transcriptional profiling by means of RNA-sequencing, chromatin immunoprecipitation (ChIP) sequencing, and assessment of the inflammasome function of primary macrophages were performed. Studies revealed a novel mechanism of genome-wide gain-of-function that dysregulated transcription of 464 genes. Mechanisms involved dysregulated noncanonical inflammasome activation caused by decreased association with transcriptional repressors, leading to increased chromatin occupancy and considerable changes in transcriptional activity, including increased expression of NLR family, pyrin domain-containing 3 protein (NLRP3) and constitutively expressed caspase-5 in macrophages. We describe a novel autoinflammatory disease with defective neutrophil function caused by a homozygous Arg219His mutation in the transcription factor C/EBPε. Mutated C/EBPε acts as a regulator of both the inflammasome and interferome, and the Arg219His mutation causes the first human monogenic neomorphic and noncanonical inflammasomopathy/immunodeficiency. The mechanism, including widely dysregulated transcription, is likely not unique for C/EBPε. Similar multiomics approaches should also be used in studying other transcription factor-associated diseases.

CEBPE (CCAAT/enhancer binding protein epsilon)

CEBPE (CCAAT/enhancer binding protein epsilon)

Genetic predisposition to B-cell acute lymphoblastic leukemia at 14q11.2 is mediated by a CEBPE promoter polymorphism

Acute lymphoblastic leukaemia (ALL) is the most common paediatric malignancy. Genome-wide association studies have shown variation at 14q11.2 influences ALL risk. We sought to decipher causal variant(s) at 14q11.2 and the mechanism of tumorigenesis. We show rs2239630 G>A resides in the promoter of the CCAT enhancer-binding protein epsilon (CEBPE) gene. The rs2239630-A risk allele is associated with increased promotor activity and CEBPE expression. Depletion of CEBPE in ALL cells reduces cell growth, correspondingly CEBPE binds to the promoters of electron transport and energy generation genes. RNA-seq in CEBPE depleted cells demonstrates CEBPE regulates the expression of genes involved in B-cell development (IL7R), apoptosis (BCL2), and methotrexate resistance (RASS4L). CEBPE regulated genes significantly overlapped in CEBPE depleted cells, ALL blasts and IGH-CEBPE translocated ALL. This suggests CEBPE regulates a similar set of genes in each, consistent with a common biological mechanism of leukemogenesis for rs2239630 associated and CEBPE translocated ALL. Finally, we map IGH-CEBPE translocation breakpoints in two cases, implicating RAG recombinase activity in their formation.

Abstract 1506: Characterization of fibronectin type III domain containing 3B (FNDC3B) as a novel fusion partner of retinoic acid receptor alpha in granulocytic differentiation

Abstract Acute promyelocytic leukemia (APL) is characterized by the promyelocytic leukemia-retinoic acid receptor alpha (PML-RARA) fusion. Recently, we identified fibronectin type III domain containing 3B (FNDC3B) as a novel RARA fusion partner resulting from the t(3;17)(q26;q21) translocation in a variant APL patient. Unlike other RARA fusion partners, FNDC3B appears as the only partner involved in granulocytic differentiation but the molecular mechanisms underlying its actions remain to be characterized. FNDC3B mRNA expression in normal and malignant hematopoietic cells was determined by quantitative RT-PCR. Transcriptional control of FNDC3B was studied by reporter gene assays, site-directed mutagenesis and chromatin immunoprecipitation (ChIP) assays. FNDC3B-regulated pathways were identified by transcriptome analyses. Using BloodSpot, we observed that FNDC3B expression was progressively up-regulated as human hematopoietic stem cells (HSCs) differentiate along the granulocytic lineage, with the highest level in terminally differentiated polymorphonuclear cells. Concordantly, we demonstrated lower expression of FNDC3B in human cord blood CD34+ HSCs than HL-60 promyelocytes. The specific involvement of FNDC3B in granulocytic differentiation was further suggested by higher FNDC3B expression and promoter activity in human promyelocytic than monocytic cell lines. Mutational analyses indicated that two putative CCAAT-enhancer binding protein epsilon (CEBPE) and one Sp1 binding sites were critical for FNDC3B promoter activity in HL-60 cells. The specific association of CEBPE and Sp1 with the FNDC3B promoter could be confirmed by ChIP assays. Knockdown of CEBPE in HL60 cells by small interfering RNA (siRNA) reduced FNDC3B expression and promoter activity. Furthermore, we observed a strong positive correlation between CEBPE and FNDC3B expression in the Cancer Genome Atlas (TCGA)-acute myeloid leukemia (AML) dataset (n=173, r=0.66, P&amp;lt;0.0001). Collectively, these findings indicate CEBPE, which is critical in terminal granulopoiesis, as an important factor controlling FNDC3B transcription in granulocytic cells. Using RNA-seq, we found that FNDC3B knockdown in NB4 promyelocytes was associated with preferential downregulation of genes encoding granule and endoplasmic reticulum (ER) structural/functional proteins. Concordantly, DAVID functional annotation of genes that are positively correlated with FNDC3B in the TCGA-AML cohort revealed enrichment of Gene Ontology terms related to ER functions. Together, these findings suggest that FNDC3B mediates its functions primarily by targeting the ER/granule pathway, which is important for granulocytic maturation. In summary, our data indicate FNDC3B as a novel CEBPE target in the myeloid transcriptional hierarchy and may provide new clues in abnormal granulopoiesis. Citation Format: Zhe Wand, Chi Keung Cheng, Terry Wong, Kin Mang Lau, Thomas S.k. Wan, Kam Tong Leung, Margaret Heung-Ling Ng. Characterization of fibronectin type III domain containing 3B (FNDC3B) as a novel fusion partner of retinoic acid receptor alpha in granulocytic differentiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1506.

Identification and Changes in the Seasonal Concentrations of Proteins Regulating the Biological Functions of Spermatozoa and Participating in Their Cytoskeleton Organization in Roe Deer (Capreolus Capreolus) Epididymides

Abstract In the case of animals characterized by a strongly marked seasonality of reproduction, extremely significant seem to be periodical fluctuations in the concentration of proteinaceous substances identified in tissues and fluids of epididymides, which take part in the maturation process of sperm cells. The aim of the present study was to compare the seasonal expression of identified proteins present in various regions of the mature roe deer (Capreolus capreolus) epididymides, which are regulating the biological functions of spermatozoa and participating in their cytoskeleton organization during the sperm maturation process. Epididymal tissue homogenates and epididymal fluids were analyzed by two-dimensional electrophoresis (2D-PAGE) and tandem mass spectrometry (MS/MS) to reveal 31 polypeptides with different biochemical functions. Moreover, among the identified polypeptides, twelve of them were similar to: alpha-enolase isoform 3; endoplasmic reticulum resident protein 29; calreticulin, calponin-1 isoform X1; transgelin; vimentin; tubulin; desmin; tropomyosin; actin; alpha-1 antitrypsin isoform X1 and 14-3-3 protein epsilon. Concentrations of the analyzed polypeptides, expressed in optical density units (ODU), differed significantly (P≤0.05) across the examined periods of the reproductive season. The highest ODU values of almost all analyzed proteins were observed during the rutting period. This variation in the identified proteins in the epididymal tissues and fluids of roe deer throughout the reproductive season could be indicative of their important roles in sperm maturation within the epididymis.

Identification of differentially regulated deceitful proteins in SH‐SY5Y cells engineered with Tet‐regulated protein expression system

Tetracycline regulated protein expression in mammalian cells is a powerful tool to predict the physiological function, cellular localization, and stability of a protein. In addition, to predict metabolic networks affected by the expression of wild-type or mutant forms of proteins, researchers generally produce a single mammalian cell clone that can express the protein of interest under tetracycline control and study the changes occurring in overall proteome before and after expression of a protein of interest. One limitation of tetracycline regulated clonal cell creation, however, is that it sometimes creates clones with changed protein levels even without the expression of the protein of interest due to the nonspecific insertion of the gene encoding the protein of interest into the genome or disruption of a metabolic pathway due to insertional silencing or activation. The aim of this study was to demonstrate the limitation of tetracycline regulated gene expression by creating clonal cell lines expressing the wild-type or the mutant forms of Fat mass and obesity-associated protein. Comparative proteome analysis of the protein extracts by two-dimensional gel electrophoresis coupled to MALDI-TOF/TOF revealed the presence of eight proteins subjected to differential regulation even in the absence of induction. The identified proteins were 14-3-3 protein Epsilon, Vimentin, Heterogeneous nuclear ribonucleoprotein K, Tubulin beta-2C chain, Heat shock protein HSP 90-alpha, Heat shock protein HSP 90-beta, Alpha-enolase, TATA-binding protein-associated factor 2N. An ultimate care should be taken to prevent reporting of deceitful proteins generated from studies utilizing tetracycline regulated gene expression systems.

A recombinant Fasciola gigantica 14-3-3 epsilon protein (rFg14-3-3e) modulates various functions of goat peripheral blood mononuclear cells

BackgroundThe molecular structure of Fasciola gigantica 14-3-3 protein has been characterized. However, the involvement of this protein in parasite pathogenesis remains elusive and its effect on the functions of innate immune cells is unknown. We report on the cloning and expression of a recombinant F. gigantica 14-3-3 epsilon protein (rFg14-3-3e), and testing its effects on specific functions of goat peripheral blood mononuclear cells (PBMCs).MethodsrFg14-3-3e protein was expressed in Pichia pastoris. Western blot and immunofluorescence assay (IFA) were used to examine the reactivity of rFg14-3-3e protein to anti-F. gigantica and anti-rFg14-3-3e antibodies, respectively. Various assays were used to investigate the stimulatory effects of the purified rFg14-3-3e protein on specific functions of goat PBMCs, including cytokine secretion, proliferation, migration, nitric oxide (NO) production, phagocytosis, and apoptotic capabilities. Potential protein interactors of rFg14-3-3e were identified by querying the databases Intact, String, BioPlex and BioGrid. A Total Energy analysis of each of the identified interaction was performed. Gene Ontology (GO) enrichment analysis was conducted using Funcassociate 3.0.ResultsSequence analysis revealed that rFg14-3-3e protein had 100% identity to 14-3-3 protein from Fasciola hepatica. Western blot analysis showed that rFg14-3-3e protein is recognized by sera from goats experimentally infected with F. gigantica and immunofluorescence staining using rat anti-rFg14-3-3e antibodies demonstrated the specific binding of rFg14-3-3e protein to the surface of goat PBMCs. rFg14-3-3e protein stimulated goat PBMCs to produce interleukin-10 (IL-10) and transforming growth factor beta (TGF-β), corresponding with low levels of IL-4 and interferon gamma (IFN-γ). Also, this recombinant protein promoted the release of NO and cell apoptosis, and inhibited the proliferation and migration of goat PBMCs and suppressed monocyte phagocytosis. Homology modelling revealed 65% identity between rFg14-3-3e and human 14-3-3 protein YWHAE. GO enrichment analysis of the interacting proteins identified terms related to apoptosis, protein binding, locomotion, hippo signalling and leukocyte and lymphocyte differentiation, supporting the experimental findings.ConclusionsOur data suggest that rFg14-3-3e protein can influence various cellular and immunological functions of goat PBMCs in vitro and may be involved in mediating F. gigantica pathogenesis. Because of its involvement in F. gigantica recognition by innate immune cells, rFg14-3-3e protein may have applications for development of diagnostics and therapeutic interventions.

17β-estradiol Changes the Human Cerebral Endothelial Cell Proteome Upon Exposure to Cyclic Stretch

Hypertension and estrogen deficiency in women have been identified as significant risk factors for cerebrovascular diseases. Hypertension causes excessive vascular stretch and contributes to the initiation of cellular injury in blood vessels while estrogen has been demonstrated to exert beneficial protective effects on the vascular system. Although the specific biological outcomes exerted by either excessive stretch or estrogen exposure are well established, the combined biochemical effects of both stimuli remain unclear. Therefore, this study was conducted for quantitative proteomics study on human cerebral microvascular endothelial cells (HCMECs) subjected to 20% “pathological” cyclic stretch for a period of 18 hour in the presence or absence of 17β-estradiol by isobaric Taqs for Relative and Absolute Quantification. The results showed that only some proteins responded to 17β-estradiol (e.g., thioredoxin reductase-1), stretch (e.g., 14- 3-3 protein epsilon or acidic leucine-rich nuclear phosphoprotein 32 family member B) and interestingly, some proteins returned to control pre-treatment levels when exposed to both (e.g., d-dopachrome decarboxylase, thrombospondin-1). In addition, HCMECs that exposed only to estrogen had a very similar proteomic profile (i.e., up-regulation of structural, cellular adhesion and proliferation proteins) as to those exposed to estrogen with 20% stretching for 18 hour, suggesting that estrogen ablated the detrimental effects by the stretch alone. These findings sheds light on the molecular mechanisms by which the cerebrovascular protective actions of estrogen on HCMEC exposed to pathological levels of cyclic stretch which could provide a platform for future research in therapeutic approach. DOI : http://dx.doi.org./10.17576/JSKM-2018-09

17β-estradiol Changes the Human Cerebral Endothelial Cell Proteome Upon Exposure to Cyclic Stretch

Hypertension and estrogen deficiency in women have been identified as significant risk factors for cerebrovascular diseases. Hypertension causes excessive vascular stretch and contributes to the initiation of cellular injury in blood vessels while estrogen has been demonstrated to exert beneficial protective effects on the vascular system. Although the specific biological outcomes exerted by either excessive stretch or estrogen exposure are well established, the combined biochemical effects of both stimuli remain unclear. Therefore, this study was conducted for quantitative proteomics study on human cerebral microvascular endothelial cells (HCMECs) subjected to 20% “pathological” cyclic stretch for a period of 18 hour in the presence or absence of 17β-estradiol by isobaric Taqs for Relative and Absolute Quantification. The results showed that only some proteins responded to 17β-estradiol (e.g., thioredoxin reductase-1), stretch (e.g., 14- 3-3 protein epsilon or acidic leucine-rich nuclear phosphoprotein 32 family member B) and interestingly, some proteins returned to control pre-treatment levels when exposed to both (e.g., d-dopachrome decarboxylase, thrombospondin-1). In addition, HCMECs that exposed only to estrogen had a very similar proteomic profile (i.e., up-regulation of structural, cellular adhesion and proliferation proteins) as to those exposed to estrogen with 20% stretching for 18 hour, suggesting that estrogen ablated the detrimental effects by the stretch alone. These findings sheds light on the molecular mechanisms by which the cerebrovascular protective actions of estrogen on HCMEC exposed to pathological levels of cyclic stretch which could provide a platform for future research in therapeutic approach. DOI : http://dx.doi.org./10.17576/JSKM-2018-09