ExoProK: A Practical Method for the Isolation of Small Extracellular Vesicles from Pleural Effusions

Extracellular vesicles (EVs) are cell-secreted, lipid membrane-enclosed nanoparticles without functional nucleus. EV is a general term that includes various subtypes of particles named microvesicles, microparticles, ectosomes or exosomes. EVs transfer RNA, DNA and protein cargo between proximal and distant cells and tissues, thus constituting an organism-wide signal transduction network. Pathological tissues secrete EVs that differ in their cargo composition compared to their healthy counterparts. The detection of biomarkers in EVs from biological fluids may aid the diagnosis of disease and/or monitor its progression in a minimally invasive manner. Among biological fluids, pleural effusions (PEs) are integrated to clinical practice, as they accompany a wide variety of lung disorders. Due to the proximity with the pleura and the lungs, PEs are expected to be especially enriched in EVs that originate from diseased tissues. However, PEs are among the least studied biofluids regarding EV-specialized isolation methods and related biomarkers. Herein, we describe a practical EV isolation method from PEs for the screening of EV RNA biomarkers in clinical routine. It is based on a Proteinase K treatment step to digest contaminants prior to standard polyethylene-glycol precipitation. The efficiency of the method was confirmed by transmission electron microscopy, nanoparticle tracking analysis and Western blot. The reliability and sensitivity of the method towards the detection of EV-enriched RNA biomarkers from multiple PEs was also demonstrated

DNP-poly(A) is a competitive inhibitor of PARN.

<p>(A). Double reciprocal plots 1/v versus 1/[substrate] for PARN activity in the presence or absence (•) of DNP-poly(A) are shown. The DNP-poly(A) concentrations are 0.1 (▪), 0.3 (⧫) and 0.9 (▴) and 1.8 (▾) mM. Representative plots of at least three independent experiments. Substrate concentrations range from 0.1–0.6 mM poly(A). (B). The slopes (<i>K</i>_M^app/<i>V</i>_max) of the double reciprocal lines are plotted versus the DNP-poly(A) concentration used to calculate the <i>K</i>_i value. The intercept of line on <i>x</i>-axis represents –<i>K</i>_i.</p

A representation of the 3D organization of the catalytic site of PARN.

<p>The RNA interacting and structurally conserved residues (Asp324, Thr325, Gly70, Gln68, Leu343, Asn288, Lys326) are shown in an electrostatic cloud, whereas the four evolutionary invariant amino acids that conformationally support the catalytic residues are shown in specefill representation (labeled as under-layer, Asp324, Thr325, Gln68, Gly70). The invariant residues that were detected in the PARN protein motifs by our phylogenetic analysis are showing below the 3D structure.</p

The role of Arg99 in the catalytic mechanism of PARN.

<p>(A) PARN - Poly(A) interactions have been calculated for both active sites. The Arg99 residues have been highlighted while they are H-bonding with the base moiety of the first poly(A) nucleotide. (B) The interaction map of poly(A) and the catalytic site of human PARN, showing the water mediated bridges of the Aspartic residue attacking the first phosphodiesteric bond, and the vital contribution of the invariant, structurally conserved His377 residue.</p

PARN phylogenetic analysis and sequence motifs.

<p>(A) Phylogenetic tree of PARN proteins. Colored boxes identify different eukaryotic groups. Bootstrap values (>50%) are shown at the nodes. The length of the tree branch reflects evolutionary distance. The scale bar at the upper left represents evolutionary distance of 0.5 amino acids per position.(B) Sequence logo of the motifs identified in PARN protein sequences. The amino acid residue numbers (according to human PARN numbering) are indicated at the top. The height of each letter is proportional to the frequency of the corresponding residue at that position, and the letters are ordered so the most frequent is on the top. The invariant residues are indicated with dots.</p

Integrated Deadenylase Genetic Association Network and Transcriptome Analysis in Thoracic Carcinomas

The poly(A) tail at the 3&prime; end of mRNAs determines their stability, translational efficiency, and fate. The shortening of the poly(A) tail, and its efficient removal, triggers the degradation of mRNAs, thus, regulating gene expression. The process is catalyzed by a family of enzymes, known as deadenylases. As the dysregulation of gene expression is a hallmark of cancer, understanding the role of deadenylases has gained additional interest. Herein, the genetic association network shows that CNOT6 and CNOT7 are the most prevalent and most interconnected nodes in the equilibrated diagram. Subsequent silencing and transcriptomic analysis identifies transcripts possibly regulated by specific deadenylases. Furthermore, several gene ontologies are enriched by common deregulated genes. Given the potential concerted action and overlapping functions of deadenylases, we examined the effect of silencing a deadenylase on the remaining ones. Our results suggest that specific deadenylases target unique subsets of mRNAs, whilst at the same time, multiple deadenylases may affect the same mRNAs with overlapping functions

Proteomic Analysis of Human Angiogenin Interactions Reveals Cytoplasmic PCNA as a Putative Binding Partner

Human Angiogenin (hAng) is a member of the ribonuclease A superfamily and a potent inducer of neovascularization. Protein interactions of hAng in the nucleus and cytoplasm of the human umbilical vein cell line EA.hy926 have been investigated by mass spectroscopy. Data are available via ProteomeXchange with identifiers PXD006583 and PXD006584. The first gel-free analysis of hAng immunoprecipitates revealed many statistically significant potential hAng-interacting proteins involved in crucial biological pathways. Surprisingly, proliferating cell nuclear antigen (PCNA), was found to be immunoprecipitated with hAng only in the cytoplasm. The hAng–PCNA interaction and colocalization in the specific cellular compartment was validated with immunoprecipitation, immunoblotting, and immunocytochemistry. The results revealed that PCNA is predominantly localized in the cytoplasm, while hAng is distributed both in the nucleus and in the cytoplasm. hAng and PCNA colocalize in the cytoplasm, suggesting that they may interact in this compartment

AtHESPERIN: a novel regulator of circadian rhythms with poly(A)-degrading activity in plants

<p>We report the identification and characterization of a novel gene, <i>AtHesperin</i> (<i>AtHESP</i>) that codes for a deadenylase in <i>Arabidopsis thaliana</i>. The gene is under circadian clock-gene regulation and has similarity to the mammalian <i>Nocturnin</i>. AtHESP can efficiently degrade poly(A) substrates exhibiting allosteric kinetics. Size exclusion chromatography and native electrophoresis coupled with kinetic analysis support that the native enzyme is oligomeric with at least 3 binding sites. Knockdown and overexpression of <i>AtHESP</i> in plant lines affects the expression and rhythmicity of the clock core oscillator genes <i>TOC1</i> and <i>CCA1</i>. This study demonstrates an evolutionary conserved poly(A)-degrading activity in plants and suggests deadenylation as a mechanism involved in the regulation of the circadian clock. A role of <i>AtHESP</i> in stress response in plants is also depicted.</p

An Integrated In Silico Approach to Design Specific Inhibitors Targeting Human Poly(A)-Specific Ribonuclease

Poly(A)-specific ribonuclease (PARN) is an exoribonuclease/deadenylase that degrades 3'-end poly(A) tails in almost all eukaryotic organisms. Much of the biochemical and structural information on PARN comes from the human enzyme. However, the existence of PARN all along the eukaryotic evolutionary ladder requires further and thorough investigation. Although the complete structure of the full-length human PARN, as well as several aspects of the catalytic mechanism still remain elusive, many previous studies indicate that PARN can be used as potent and promising anti-cancer target. In the present study, we attempt to complement the existing structural information on PARN with in-depth bioinformatics analyses, in order to get a hologram of the molecular evolution of PARNs active site. In an effort to draw an outline, which allows specific drug design targeting PARN, an unequivocally specific platform was designed for the development of selective modulators focusing on the unique structural and catalytic features of the enzyme. Extensive phylogenetic analysis based on all the publicly available genomes indicated a broad distribution for PARN across eukaryotic species and revealed structurally important amino acids which could be assigned as potentially strong contributors to the regulation of the catalytic mechanism of PARN. Based on the above, we propose a comprehensive in silico model for the PARN's catalytic mechanism and moreover, we developed a 3D pharmacophore model, which was subsequently used for the introduction of DNP-poly(A) amphipathic substrate analog as a potential inhibitor of PARN. Indeed, biochemical analysis revealed that DNP-poly(A) inhibits PARN competitively. Our approach provides an efficient integrated platform for the rational design of pharmacophore models as well as novel modulators of PARN with therapeutic potential. Citation: Vlachakis D, Pavlopoulou A, Tsiliki G, Komiotis D, Stathopoulos C, et al. (2012) An Integrated In Silico Approach to Design Specific Inhibitors Targeting Human Poly(A)-Specific Ribonuclease. PLoS ONE 7(12): e51113. doi:10.1371/journal.pone.005111