Binding Partners of Parvulin Proteins using the High-Throughput Screening Methods such as Yeast two-hybrid and Phage Display

Peptidyl prolyl cis/trans isomerases (PPIases) are the enzymes that increase the rate of isomerization of the peptide bond N-terminal to the proline substrate. Parvulin14 (Par14) and its isoform Par17 belong to the Parvulin family which is the third family of PPIases. Par14 was shown to bind the DNA. Par14 was also proposed to be a part from the pre-ribosomal ribonucleoprotien complexes and an RNA processing factor that is involved in ribosome biogenesis. The longer isoform Par17 was shown to be expressed only in the Hominidae, and is targeted to the mitochondria. In order to find binding partners for Par14/17 (peptides or proteins), we applied the high through-put screening methods, the yeast two-hybrid (Y2H) and the phage display (Ph.D.). In our Y2H study we show a target-unrelated protein that may confuse a number of Y2H results. In our Ph.D. screening against PinA of Cenarchaeum symbiosum we selected a peptide that binds PinA with low affinity; however we used this peptide to map PinA putative PPIase active site, and to show a flexible region of PinA. The flexibility of this region is probably a general feature of the peptidyl prolyl cis/trans isomerization. Furthermore, we panned 7 and 12-mer peptide libraries against Par17. One consensus sequence was enriched from both libraries, XHSXVHØ, where X can be any amino acid and Ø is a hydrophobic amino acid. We demonstrate the binding of this motif to Par14/17 with phage ELISA and NMR spectroscopy where we could show that this motif is binding to the PPIase domain of Par14/17. Moreover, using these peptides we map the PPIase active site of Par14/17. Our peptides can be used to design peptides to study the PPIase activity of Par14/17, and to elucidate the motif that Par14/17 recognizes in vivo.Peptidyl-Prolyl-cis/trans-Isomerasen (PPIasen) sind Enzyme, die cis/trans-Isomerisierung von Peptidyl-Prolyl-Bindungen (Xaa-Pro-Bindungen) katalysieren. Parvulin 14 (Par14) und seine Isoform Par17 gehören zu der Familie der Parvuline, einer Untergruppe der PPIasen. Für Par14 wurde gezeigt, dass es an DNA bindet. In einer anderen Studie wurde gezeigt, dass Par14 Teil des preribosomalen Ribonukleoprotein-Komplexes ist und ein RNA-Prozessierungsfaktor sei, welcher in der Ribosomenbiogenese involviert ist. Par17 kommt nur in Hominidae vor und ist in den Mitochondrien lokalisiert. Um Bindungspartner für Par14/17 (Peptide oder Proteine) zu finden, wurden Hochdurchsatz-Screening-Verfahren, das Yeast-two-Hybrid-System (Y2H) und das Phagen-Display (Ph.D.) verwendet. In der Y2H-Studie wurde ein target-unrelated-protein (TUP) gefunden. Im Ph.D. Screening gegen PinA von Cenarchaeum symbiosum wurde ein Peptid gefunden, das PinA mit niedriger Affinität bindet. Mithilfe des Peptids konnte ein vermutliches aktives Zentrum der PPIase PinA zugeordnet werden. Ebenfalls wurden eine 7 und 12-mer Peptidbibliothek gegen Par17 entwickelt. Die Konsensus-Sequenz XHSXVHØ wurde aus beiden Bibliotheken angereichert, wobei X eine beliebige Aminosäure und Ø eine hydrophobe Aminosäure darstellt. Die Bindung dieses Motivs an Par14/17 wurde über Phagen-ELISA und NMR-Spektroskopie untersucht, wobei gezeigt werden konnte, dass dieses Motiv an die Par14/17 PPIase Domäne bindet. Mithilfe dieser Peptide wurde das putative aktive Zentrum von Par14/17 in den NMR-Strukturen zugeordnet. Desweiteren können besagte Peptide zukünftig dazu verwendet werden, potentielle Bindungspartner von Par14/17 zu finden

CYTOCHROME P450 CYP1B1*2 GENE AND ITS ASSOCIATION WITH T2D IN TABUK POPULATION, NORTHWESTERN REGION OF SAUDI ARABIA

Objective: Cytochrome P450 1B1 (CYP1B1) is involved in the activation of procarcinogens and steroid metabolism. Genetic variants of CYP1B1are associated with altered catalytic activity and disease phenotypes. The purpose of this study was to investigate the role of CYP1B1 (rs1056827) polymorphism in inducing T2D.Methods: This cross-sectional study enrolled 113 subjects of T2D and 120 controls. DNA was isolated from blood. Genotyping of the rs1056827 wasdone by allele-specific polymerase chain reaction. The frequency of alleles and genotype distribution was compared in T2D cases and healthy controls.Statistical analysis was performed with SPSS, Chi-square, and Fisher exact test. Hardy-Weinberg equilibrium was tested by a χ2 test. The associations between rs1056827 variant genotypes and T2D were estimated by computing the odds ratios and their 95% confidence intervals (CI) from univariate and multivariate logistic regression analysis.Results: A significant association of rs1056827 was found between T2D cases and controls (p<0.0001). When GG genotype was compared with GT genotype a significant association was found with odd ration (OD)0.24 (95% CI: (0.131–0.452) and risk ratio (RR) 0.45 (0.30–0.67) times the risk of T2D heterozygous with the G/T allele (p≤0.0002). In a comparison of GG homozygous with the TT homozygous, there was no significant association with the OD 0.38 (95% CI: (0.02–6.51) RR 0.55(0.13–2.35), p<0.49. When G allele was compared with the T allele a highly significant association with OD 0.54 (95% [CI]: (0.37–0.80) RR 0.75(0.630–0.897) < p≤0.003 suggesting a possible dominant effect of this polymorphism on T2D risk.Conclusion: This result suggests a significant association between rs1056827G>T polymorphism and T2D. This finding is limited due to the smaller sample size and can be validated by large sample size studies

Role of non-coding RNA networks in leukemia progression, metastasis and drug resistance.

Early-stage detection of leukemia is a critical determinant for successful treatment of the disease and can increase the survival rate of leukemia patients. The factors limiting the current screening approaches to leukemia include low sensitivity and specificity, high costs, and a low participation rate. An approach based on novel and innovative biomarkers with high accuracy from peripheral blood offers a comfortable and appealing alternative to patients, potentially leading to a higher participation rate.Recently, non-coding RNAs due to their involvement in vital oncogenic processes such as differentiation, proliferation, migration, angiogenesis and apoptosis have attracted much attention as potential diagnostic and prognostic biomarkers in leukemia. Emerging lines of evidence have shown that the mutational spectrum and dysregulated expression of non-coding RNA genes are closely associated with the development and progression of various cancers, including leukemia. In this review, we highlight the expression and functional roles of different types of non-coding RNAs in leukemia and discuss their potential clinical applications as diagnostic or prognostic biomarkers and therapeutic targets

Long non-coding RNAs modulate tumor microenvironment to promote metastasis: novel avenue for therapeutic intervention

Cancer is a devastating disease and the primary cause of morbidity and mortality worldwide, with cancer metastasis responsible for 90% of cancer-related deaths. Cancer metastasis is a multistep process characterized by spreading of cancer cells from the primary tumor and acquiring molecular and phenotypic changes that enable them to expand and colonize in distant organs. Despite recent advancements, the underlying molecular mechanism(s) of cancer metastasis is limited and requires further exploration. In addition to genetic alterations, epigenetic changes have been demonstrated to play an important role in the development of cancer metastasis. Long non-coding RNAs (lncRNAs) are considered one of the most critical epigenetic regulators. By regulating signaling pathways and acting as decoys, guides, and scaffolds, they modulate key molecules in every step of cancer metastasis such as dissemination of carcinoma cells, intravascular transit, and metastatic colonization. Gaining a good knowledge of the detailed molecular basis underlying lncRNAs regulating cancer metastasis may provide previously unknown therapeutic and diagnostic lncRNAs for patients with metastatic disease. In this review, we concentrate on the molecular mechanisms underlying lncRNAs in the regulation of cancer metastasis, the cross-talk with metabolic reprogramming, modulating cancer cell anoikis resistance, influencing metastatic microenvironment, and the interaction with pre-metastatic niche formation. In addition, we also discuss the clinical utility and therapeutic potential of lncRNAs for cancer treatment. Finally, we also represent areas for future research in this rapidly developing field

Determination of Blood Glucose, Total Protein, Certain Minerals, and Triiodothyronine during Late Pregnancy and Postpartum Periods in Crossbred Dairy Cows

The late pregnancy (3rd trimester) and the postpartum period (PPP) (calving date or day zero to day 45) are very critical periods for the fertility and production in dairy cows. This study was designed to investigate blood glucose, total protein (TP), calcium (Ca), phosphorus (P), magnesium (Mg), iron (Fe), and triiodothyronine (T3) during late pregnancy and the PPP. Twenty-seven apparently healthy multiparous crossbred dairy cows (Friesian × Kenana) were included in this study. The cows were randomly allocated into three groups: group A (n = 10), cows with late pregnancy, group B (n = 7), cows in the PPP, and group C (n = 10), nonpregnant cows as control. One-way ANOVA was used to analyze the data. The results of this study showed that blood glucose was higher in late pregnancy and the PPP than in nonpregnant cows. The TP was significantly lower in late pregnant cows than during the PPP and in nonpregnant cows. Ca, P, and Mg were not significantly different between periods. Serum Fe and T3 were significantly lower during the PPP than that in late pregnant and nonpregnant cows. The results can provide indications of the nutritional status of dairy cows and a diagnostic tool to avoid the metabolic disorders that may occur during late pregnancy and the PPP

LDLR Gene Polymorphisms (rs5925 and rs1529729) Are Associated with Susceptibility to Coronary Artery Disease in a South Indian Population

Cardiovascular diseases (CVD) are a major cause of death in India and worldwide. Atherosclerosis is caused by the interaction of environmental and genetic factors. Hypercholesterolemia is an example of a classical risk factor for CVD. The low-density lipoprotein receptor (LDLR) is one of the regulating mechanisms the liver uses for cholesterol homeostasis. Gene variations in the LDLR have been reported to cause hypercholesterolemia and consequently CVD. We investigated the association of polymorphisms in the LDLR (rs5925 and rs1529729) with coronary artery disease (CAD) in 200 coronary artery disease patients and 200 matched healthy controls using allele-specific PCR (AS-PCR). The results indicated that the CT genotype of the rs1529729 polymorphism was associated a decreased susceptibility to CAD with an odds ratio (OR) = 0.42 (95% confidence interval (CI), 0.23–0.77), risk ratio (RR) = 0.59 (0.39–0.89), P = 0.0047. The TT genotype of the rs1529729 polymorphism was also associated with decreased susceptibility to CAD with an OR = 0.19 (95% CI, 0.076–0.47), RR = 0.57 (0.47–0.69), P = 0.0003. The GA genotype of the rs5925 polymorphism was associated with decreased susceptibility to CAD with an OR = 0.45 (95% CI, 0.27–0.75), RR = 0.65 (0.47–0.88), P = 0.002. We concluded that the CT and TT genotypes of the rs1529729 polymorphism and the GA genotype of the rs5925 polymorphism are probably associated with decreased susceptibility to CAD. The simplicity of AS-PCR makes it particularly suitable for the rapid, large-scale screening of gene variabilities in the LDLR. AS-PCR could provide significant benefits in clinical applications with its ability to amplify a lower quantity of samples in a cost-saving manner. Nevertheless, these findings need to be validated in well-designed studies with larger sample sizes and in different populations

Structure and Dynamics of the First Archaeal Parvulin Reveal a New Functionally Important Loop in Parvulin-type Prolyl Isomerases*

Parvulins are a group of peptidyl-prolyl isomerases (PPIases) responsible for important biological processes in all kingdoms of life. The PinA protein from the psychrophilic archaeon Cenarchaeum symbiosum is a parvulin-like PPIase. Due to its striking similarity to the human parvulins Pin1 and Par14, PinA constitutes an interesting subject for structural and functional studies. Here, we present the first high resolution NMR structure of an archaeal parvulin, PinA, based on 1798 conformational restraints. Structure calculation yields an ensemble of 20 convergent low energy structures with a backbone r.m.s.d. value of 0.6 Å within the secondary structure elements. The overall fold of PinA comprises the β-α3-β-α-β2 fold typical for all parvulin structures known so far, but with helix III being a short 310-helix. A detailed comparison of this high resolution structure of the first archaeal PinA protein with bacterial and eukaryotic parvulin PPIase structures reveals an atypically large catalytic binding site. This feature provides an explanation for cold-adapted protein function. Moreover, the residues in and around 310-helix III exhibit strong intramolecular dynamics on a microsecond to millisecond timescale and display structural heterogeneity within the NMR ensemble. A putative peptide ligand was found for PinA by phage display and was used for 1H-15N-HSQC titrations. Again, the flexible region around 310-helix III as well as residues of the peptide binding pocket showed the strongest chemical shift perturbations upon peptide binding. The local flexibility of this region also was modulated by ligand binding. A glycine and two positively charged residues are conserved in most parvulin proteins in this flexible loop region, which may be of general functional importance for parvulin-type PPIases

Potential Impact of MicroRNA Gene Polymorphisms in the Pathogenesis of Diabetes and Atherosclerotic Cardiovascular Disease

MicroRNAs (miRNAs) are endogenous, small (18–23 nucleotides), non-coding RNA molecules. They regulate the posttranscriptional expression of their target genes. MiRNAs control vital physiological processes such as metabolism, development, differentiation, cell cycle and apoptosis. The control of the gene expression by miRNAs requires efficient binding between the miRNA and their target mRNAs. Genome-wide association studies (GWASs) have suggested the association of single-nucleotide polymorphisms (SNPs) with certain diseases in various populations. Gene polymorphisms of miRNA target sites have been implicated in diseases such as cancers, diabetes, cardiovascular and Parkinson’s disease. Likewise, gene polymorphisms of miRNAs have been reported to be associated with diseases. In this review, we discuss the SNPs in miRNA genes that have been associated with diabetes and atherosclerotic cardiovascular disease in different populations. We also discuss briefly the potential underlining mechanisms through which these SNPs increase the risk of developing these diseases

Reply to Comment: Evaluation of the Association of Omentin 1 rs2274907 A > T and rs2274908 G > A Gene Polymorphisms with Coronary Artery Disease in Indian Population: A Case–Control Study

Coronary artery disease (CAD) is a major cause of death all over the world. CAD is caused by atherosclerosis which is induced by the interaction of genetic factors and environmental factors. Genome-wide association studies have revealed the association of certain gene polymorphisms with susceptibility to CAD. Omentin 1 is an adipokine secreted by the visceral adipose tissues and has been reported to have anti-inflammatory, cardioprotective, and enhances insulin sensitivity. In this study, we examined the role of omentin-1 common single nucleotide polymorphisms (SNPs) (rs2274907 A > T and rs2274908 G > A) in CAD. We conclude that the AT genotype and the T allele of the rs2274907 A > T is associated with Cad in the south Indian population. Our results indicated that the rs2274907 SNP may be associated with CAD in this population. This finding needs further validation in well-designed and large-sample size studies before being introduced in clinical settings