PLOS ONE: [sortOrder=DATE_NEWEST_FIRST, from=editorLink, sort=Relevance, q=editor:"Vladimir N Uversky"]PLOShttps://journals.plos.org/plosone/webmaster@plos.orgaccelerating the publication of peer-reviewed sciencehttps://journals.plos.org/plosone/search/feed/atom?sortOrder=DATE_NEWEST_FIRST&unformattedQuery=editor:%22Vladimir%20N%20Uversky%22&from=editorLink&sort=RelevanceAll PLOS articles are Open Access.https://journals.plos.org/plosone/resource/img/favicon.icohttps://journals.plos.org/plosone/resource/img/favicon.ico2024-03-28T18:07:09ZGlycation of H1 Histone by 3-Deoxyglucosone: Effects on Protein Structure and Generation of Different Advanced Glycation End ProductsJalaluddin Mohammad AshrafGulam RabbaniSaheem AhmadQambar HasanRizwan Hasan KhanKhursheed AlamInho Choi10.1371/journal.pone.01306302015-06-29T14:00:00Z2015-06-29T14:00:00Z<p>by Jalaluddin Mohammad Ashraf, Gulam Rabbani, Saheem Ahmad, Qambar Hasan, Rizwan Hasan Khan, Khursheed Alam, Inho Choi</p>
Advanced glycation end products (AGEs) culminate from the non-enzymatic reaction between a free carbonyl group of a reducing sugar and free amino group of proteins. 3-deoxyglucosone (3-DG) is one of the dicarbonyl species that rapidly forms several protein-AGE complexes that are believed to be involved in the pathogenesis of several diseases, particularly diabetic complications. In this study, the generation of AGEs (N<sup>ε</sup>-carboxymethyl lysine and pentosidine) by 3-DG in H1 histone protein was characterized by evaluating extent of side chain modification (lysine and arginine) and formation of Amadori products as well as carbonyl contents using several physicochemical techniques. Results strongly suggested that 3-DG is a potent glycating agent that forms various intermediates and AGEs during glycation reactions and affects the secondary structure of the H1 protein. Structural changes and AGE formation may influence the function of H1 histone and compromise chromatin structures in cases of secondary diabetic complications.The Dimerization State of the Mammalian High Mobility Group Protein AT-Hook 2 (HMGA2)Lorraine FrostMaria A. M. BaezChristopher HarrilalAlyssa GarabedianFrancisco Fernandez-LimaFenfei Leng10.1371/journal.pone.01304782015-06-26T14:00:00Z2015-06-26T14:00:00Z<p>by Lorraine Frost, Maria A. M. Baez, Christopher Harrilal, Alyssa Garabedian, Francisco Fernandez-Lima, Fenfei Leng</p>
The mammalian high mobility group protein AT-hook 2 (HMGA2) is a chromosomal architectural transcription factor involved in cell transformation and oncogenesis. It consists of three positively charged “AT-hooks” and a negatively charged C-terminus. Sequence analyses, circular dichroism experiments, and gel-filtration studies showed that HMGA2, in the native state, does not have a defined secondary or tertiary structure. Surprisingly, using combined approaches of 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) chemical cross-linking, analytical ultracentrifugation, fluorescence resonance energy transfer (FRET), and mass spectrometry, we discovered that HMGA2 is capable of self-associating into homodimers in aqueous buffer solution. Our results showed that electrostatic interactions between the positively charged “AT-hooks” and the negatively charged C-terminus greatly contribute to the homodimer formation.Isolation of High-Purity Extracellular Vesicles by Extracting Proteins Using Aqueous Two-Phase SystemJongmin KimHyunwoo ShinJiyoon KimJunho KimJaesung Park10.1371/journal.pone.01297602015-06-19T14:00:00Z2015-06-19T14:00:00Z<p>by Jongmin Kim, Hyunwoo Shin, Jiyoon Kim, Junho Kim, Jaesung Park</p>
We present a simple and rapid method to isolate extracellular vesicles (EVs) by using a polyethylene glycol/dextran aqueous two-phase system (ATPS). This system isolated more than ~75% of melanoma-derived EVs from a mixture of EVs and serum proteins. To increase the purity of EVs, a batch procedure was combined as additional steps to remove protein contaminants, and removed more than ~95% of the protein contaminants. We also performed RT-PCR and western blotting to verify the diagnostic applicability of the isolated EVs, and detected mRNA derived from melanoma cells and CD81 in isolated EVs.Evolution of the SH3 Domain Specificity Landscape in YeastsErik VerschuerenMatthias SpiessAreti GkourtsaTeja AvulaChristiane LandgrafVictor Tapia MancillaAline HuberRudolf VolkmerBarbara WinsorLuis SerranoFrans HochstenbachBen Distel10.1371/journal.pone.01292292015-06-11T14:00:00Z2015-06-11T14:00:00Z<p>by Erik Verschueren, Matthias Spiess, Areti Gkourtsa, Teja Avula, Christiane Landgraf, Victor Tapia Mancilla, Aline Huber, Rudolf Volkmer, Barbara Winsor, Luis Serrano, Frans Hochstenbach, Ben Distel</p>
To explore the conservation of Src homology 3 (SH3) domain-mediated networks in evolution, we compared the specificity landscape of these domains among four yeast species, <i>Saccharomyces cerevisiae</i>, <i>Ashbya gossypii</i>, <i>Candida albicans</i>, and <i>Schizosaccharomyces pombe</i>, encompassing 400 million years of evolution. We first aligned and catalogued the families of SH3-containing proteins in these four species to determine the relationships between homologous domains. Then, we tagged and purified all soluble SH3 domains (82 in total) to perform a quantitative peptide assay (SPOT) for each SH3 domain. All SPOT readouts were hierarchically clustered and we observed that the organization of the SH3 specificity landscape in three distinct profile classes remains conserved across these four yeast species. We also produced a specificity profile for each SH3 domain from manually aligned top SPOT hits and compared the within-family binding motif consensus. This analysis revealed a striking example of binding motif divergence in a <i>C</i>. <i>albicans</i> Rvs167 paralog, which cannot be explained by overall SH3 sequence or interface residue divergence, and we validated this specificity change with a yeast two-hybrid (Y2H) assay. In addition, we show that position-weighted matrices (PWM) compiled from SPOT assays can be used for binding motif screening in potential binding partners and present cases where motifs are either conserved or lost among homologous SH3 interacting proteins. Finally, by comparing pairwise SH3 sequence identity to binding profile correlation we show that for ~75% of all analyzed families the SH3 specificity profile was remarkably conserved over a large evolutionary distance. Thus, a high sequence identity within an SH3 domain family predicts conserved binding specificity, whereas divergence in sequence identity often coincided with a change in binding specificity within this family. As such, our results are important for future studies aimed at unraveling complex specificity networks of peptide recognition domains in higher eukaryotes, including mammals.Phosphorylation of PPARγ Affects the Collective Motions of the PPARγ-RXRα-DNA ComplexJustin A. LemkulStephanie N. LewisJosep Bassaganya-RieraDavid R. Bevan10.1371/journal.pone.01239842015-05-08T14:00:00Z2015-05-08T14:00:00Z<p>by Justin A. Lemkul, Stephanie N. Lewis, Josep Bassaganya-Riera, David R. Bevan</p>
Peroxisome-proliferator activated receptor-γ (PPARγ) is a nuclear hormone receptor that forms a heterodimeric complex with retinoid X receptor-α (RXRα) to regulate transcription of genes involved in fatty acid storage and glucose metabolism. PPARγ is a target for pharmaceutical intervention in type 2 diabetes, and insight into interactions between PPARγ, RXRα, and DNA is of interest in understanding the function and regulation of this complex. Phosphorylation of PPARγ by cyclin-dependent kinase 5 (Cdk5) has been shown to dysregulate the expression of metabolic regulation genes, an effect that is counteracted by PPARγ ligands. We applied molecular dynamics (MD) simulations to study the relationship between the ligand-binding domains of PPARγ and RXRα with their respective DNA-binding domains. Our results reveal that phosphorylation alters collective motions within the PPARγ-RXRα complex that affect the LBD-LBD dimerization interface and the AF-2 coactivator binding region of PPARγ.Amyloidogenic Propensity of a Natural Variant of Human Apolipoprotein A-I: Stability and Interaction with LigandsSilvana A. RosúOmar J. RimoldiEduardo D. PrietoLucrecia M. CurtoJosé M. DelfinoNahuel A. RamellaM. Alejandra Tricerri10.1371/journal.pone.01249462015-05-07T14:00:00Z2015-05-07T14:00:00Z<p>by Silvana A. Rosú, Omar J. Rimoldi, Eduardo D. Prieto, Lucrecia M. Curto, José M. Delfino, Nahuel A. Ramella, M. Alejandra Tricerri</p>
A number of naturally occurring mutations of human apolipoprotein A-I (apoA-I) have been associated with hereditary amyloidoses. The molecular mechanisms involved in amyloid-associated pathology remain largely unknown. Here we examined the effects of the Arg173Pro point mutation in apoA-I on the structure, stability, and aggregation propensity, as well as on the ability to bind to putative ligands. Our results indicate that the mutation induces a drastic loss of stability, and a lower efficiency to bind to phospholipid vesicles at physiological pH, which could determine the observed higher tendency to aggregate as pro-amyloidogenic complexes. Incubation under acidic conditions does not seem to induce significant desestabilization or aggregation tendency, neither does it contribute to the binding of the mutant to sodium dodecyl sulfate. While the binding to this detergent is higher for the mutant as compared to wt apoA-I, the interaction of the Arg173Pro variant with heparin depends on pH, being lower at pH 5.0 and higher than wt under physiological pH conditions. We suggest that binding to ligands as heparin or other glycosaminoglycans could be key events tuning the fine details of the interaction of apoA-I variants with the micro-environment, and probably eliciting the toxicity of these variants in hereditary amyloidoses.Glutathione S-Transferase (GST) Gene Diversity in the Crustacean <i>Calanus finmarchicus</i> – Contributors to Cellular DetoxificationVittoria RoncalliMatthew C. CieslakYale PassamaneckAndrew E. ChristiePetra H. Lenz10.1371/journal.pone.01233222015-05-06T14:00:00Z2015-05-06T14:00:00Z<p>by Vittoria Roncalli, Matthew C. Cieslak, Yale Passamaneck, Andrew E. Christie, Petra H. Lenz</p>
Detoxification is a fundamental cellular stress defense mechanism, which allows an organism to survive or even thrive in the presence of environmental toxins and/or pollutants. The glutathione S-transferase (GST) superfamily is a set of enzymes involved in the detoxification process. This highly diverse protein superfamily is characterized by multiple gene duplications, with over 40 GST genes reported in some insects. However, less is known about the GST superfamily in marine organisms, including crustaceans. The availability of two <i>de novo</i> transcriptomes for the copepod, <i>Calanus finmarchicus</i>, provided an opportunity for an in depth study of the GST superfamily in a marine crustacean. The transcriptomes were searched for putative GST-encoding transcripts using known GST proteins from three arthropods as queries. The identified transcripts were then translated into proteins, analyzed for structural domains, and annotated using reciprocal BLAST analysis. Mining the two transcriptomes yielded a total of 41 predicted GST proteins belonging to the cytosolic, mitochondrial or microsomal classes. Phylogenetic analysis of the cytosolic GSTs validated their annotation into six different subclasses. The predicted proteins are likely to represent the products of distinct genes, suggesting that the diversity of GSTs in <i>C</i>. <i>finmarchicus</i> exceeds or rivals that described for insects. Analysis of relative gene expression in different developmental stages indicated low levels of GST expression in embryos, and relatively high expression in late copepodites and adult females for several cytosolic GSTs. A diverse diet and complex life history are factors that might be driving the multiplicity of GSTs in <i>C</i>. <i>finmarchicus</i>, as this copepod is commonly exposed to a variety of natural toxins. Hence, diversity in detoxification pathway proteins may well be key to their survival.Crystal Structure of Fad35R from <i>Mycobacterium tuberculosis</i> H37Rv in the Apo-StateAppu Kumar SinghBabu ManjasettyBalasubramani GLSukirte KoulAbhishek KaushikMary Krishna EkkaVijay SinghS. Kumaran10.1371/journal.pone.01243332015-05-04T14:00:00Z2015-05-04T14:00:00Z<p>by Appu Kumar Singh, Babu Manjasetty, Balasubramani GL, Sukirte Koul, Abhishek Kaushik, Mary Krishna Ekka, Vijay Singh, S. Kumaran</p>
Fad35R from <i>Mycobacterium tuberculosis</i> binds to the promoter site of Fad35 operon and its DNA binding activities are reduced in the presence of tetracycline and palmitoyl-CoA. We resolved the crystal structure of Fad35R using single-wavelength anomalous diffraction method (SAD). Fad35R comprises canonical DNA binding domain (DBD) and ligand binding domain (LBD), but displays several distinct structural features. Two recognition helices of two monomers in the homodimer are separated by ~ 48 Å and two core triangle-shaped ligand binding cavities are well exposed to solvent. Structural comparison with DesT and QacR structures suggests that ligand binding-induced movement of α7, which adopts a straight conformation in the Fad35R, may be crucial to switch the conformational states between repressive and derepressive forms. Two DBDs are packed asymmetrically, creating an alternative dimer interface which coincides with the possible tetramer interface that connects the two canonical dimers. Quaternary state of alternative dimer mimics a closed-state structure in which two recognition helices are distanced at ~ 35 Å and ligand binding pockets are inaccessible. Results of biophysical studies indicate that Fad35R has the propensity to oligomerize in solution in the presence of tetracycline. We present the first structure of a FadR homologue from mycobacterium and the structure reveals DNA and ligand binding features of Fad35R and also provides a view on alternative quaternary states that mimic open and closed forms of the regulator.Molecular Characterization of the Llamas (<i>Lama glama</i>) Casein Cluster Genes Transcripts (<i>CSN1S1</i>, <i>CSN2</i>, <i>CSN1S2</i>, <i>CSN3</i>) and Regulatory RegionsAlfredo PauciulloGeorg Erhardt10.1371/journal.pone.01249632015-04-29T14:00:00Z2015-04-29T14:00:00Z<p>by Alfredo Pauciullo, Georg Erhardt</p>
In the present paper, we report for the first time the characterization of llama (<i>Lama glama</i>) caseins at transcriptomic and genetic level. A total of 288 casein clones transcripts were analysed from two lactating llamas. The most represented mRNA populations were those correctly assembled (85.07%) and they encoded for mature proteins of 215, 217, 187 and 162 amino acids respectively for the <i>CSN1S1</i>, <i>CSN2</i>, <i>CSN1S2</i> and <i>CSN3</i> genes. The exonic subdivision evidenced a structure made of 21, 9, 17 and 6 exons for the αs1-, β-, αs2- and κ-casein genes respectively. Exon skipping and duplication events were evidenced. Two variants A and B were identified in the αs1-casein gene as result of the alternative out-splicing of the exon 18. An additional exon coding for a novel esapeptide was found to be cryptic in the κ-casein gene, whereas one extra exon was found in the αs2-casein gene by the comparison with the <i>Camelus dromedaries</i> sequence. A total of 28 putative phosphorylated motifs highlighted a complex heterogeneity and a potential variable degree of post-translational modifications. Ninety-six polymorphic sites were found through the comparison of the lama casein cDNAs with the homologous camel sequences, whereas the first description and characterization of the 5’- and 3’-regulatory regions allowed to identify the main putative consensus sequences involved in the casein genes expression, thus opening the way to new investigations -so far- never achieved in this species.A Functional Dissection of PTEN N-Terminus: Implications in PTEN Subcellular Targeting and Tumor Suppressor ActivityAnabel GilIsabel Rodríguez-EscuderoMiriam StumpfMaría MolinaVíctor J. CidRafael Pulido10.1371/journal.pone.01192872015-04-15T14:00:00Z2015-04-15T14:00:00Z<p>by Anabel Gil, Isabel Rodríguez-Escudero, Miriam Stumpf, María Molina, Víctor J. Cid, Rafael Pulido</p>
Spatial regulation of the tumor suppressor PTEN is exerted through alternative plasma membrane, cytoplasmic, and nuclear subcellular locations. The N-terminal region of PTEN is important for the control of PTEN subcellular localization and function. It contains both an active nuclear localization signal (NLS) and an overlapping PIP2-binding motif (PBM) involved in plasma membrane targeting. We report a comprehensive mutational and functional analysis of the PTEN N-terminus, including a panel of tumor-related mutations at this region. Nuclear/cytoplasmic partitioning in mammalian cells and PIP3 phosphatase assays in reconstituted <i>S</i>. <i>cerevisiae</i> defined categories of PTEN N-terminal mutations with distinct PIP3 phosphatase and nuclear accumulation properties. Noticeably, most tumor-related mutations that lost PIP3 phosphatase activity also displayed impaired nuclear localization. Cell proliferation and soft-agar colony formation analysis in mammalian cells of mutations with distinctive nuclear accumulation and catalytic activity patterns suggested a contribution of both properties to PTEN tumor suppressor activity. Our functional dissection of the PTEN N-terminus provides the basis for a systematic analysis of tumor-related and experimentally engineered PTEN mutations.Tailor-Made Ezrin Actin Binding Domain to Probe Its Interaction with Actin <i>In-Vitro</i>Rohini ShrivastavaDarius KösterSheetal KalmeSatyajit MayorMuniasamy Neerathilingam10.1371/journal.pone.01234282015-04-10T14:00:00Z2015-04-10T14:00:00Z<p>by Rohini Shrivastava, Darius Köster, Sheetal Kalme, Satyajit Mayor, Muniasamy Neerathilingam</p>
Ezrin, a member of the ERM (Ezrin/Radixin/Moesin) protein family, is an Actin-plasma membrane linker protein mediating cellular integrity and function. <i>In-vivo</i> study of such interactions is a complex task due to the presence of a large number of endogenous binding partners for both Ezrin and Actin. Further, C-terminal actin binding capacity of the full length Ezrin is naturally shielded by its N-terminal, and only rendered active in the presence of Phosphatidylinositol bisphosphate (PIP2) or phosphorylation at the C-terminal threonine. Here, we demonstrate a strategy for the design, expression and purification of constructs, combining the Ezrin C-terminal actin binding domain, with functional elements such as fusion tags and fluorescence tags to facilitate purification and fluorescence microscopy based studies. For the first time, internal His tag was employed for purification of Ezrin actin binding domain based on <i>in-silico</i> modeling. The functionality (Ezrin-actin interaction) of these constructs was successfully demonstrated by using Total Internal Reflection Fluorescence Microscopy. This design can be extended to other members of the ERM family as well.The Dynamic Conformational Cycle of the Group I Chaperonin C-Termini Revealed via Molecular Dynamics SimulationKevin M. DaltonJudith FrydmanVijay S. Pande10.1371/journal.pone.01177242015-03-30T14:00:00Z2015-03-30T14:00:00Z<p>by Kevin M. Dalton, Judith Frydman, Vijay S. Pande</p>
Chaperonins are large ring shaped oligomers that facilitate protein folding by encapsulation within a central cavity. All chaperonins possess flexible C-termini which protrude from the equatorial domain of each subunit into the central cavity. Biochemical evidence suggests that the termini play an important role in the allosteric regulation of the ATPase cycle, in substrate folding and in complex assembly and stability. Despite the tremendous wealth of structural data available for numerous orthologous chaperonins, little structural information is available regarding the residues within the C-terminus. Herein, molecular dynamics simulations are presented which localize the termini throughout the nucleotide cycle of the group I chaperonin, GroE, from Escherichia coli. The simulation results predict that the termini undergo a heretofore unappreciated conformational cycle which is coupled to the nucleotide state of the enzyme. As such, these results have profound implications for the mechanism by which GroE utilizes nucleotide and folds client proteins.Autonomously Folding Protein Fragments Reveal Differences in the Energy Landscapes of Homologous RNases HLaura E. RosenSusan Marqusee10.1371/journal.pone.01196402015-03-24T14:00:00Z2015-03-24T14:00:00Z<p>by Laura E. Rosen, Susan Marqusee</p>
An important approach to understanding how a protein sequence encodes its energy landscape is to compare proteins with different sequences that fold to the same general native structure. In this work, we compare <i>E</i>. <i>coli</i> and <i>T</i>. <i>thermophilus</i> homologs of the protein RNase H. Using protein fragments, we create equilibrium mimics of two different potential partially-folded intermediates (I<sub>core </sub>and I<sub>core+1</sub>) hypothesized to be present on the energy landscapes of these two proteins. We observe that both <i>T</i>. <i>thermophilus</i> RNase H (ttRNH) fragments are folded and have distinct stabilities, indicating that both regions are capable of autonomous folding and that both intermediates are present as local minima on the ttRNH energy landscape. In contrast, the two <i>E</i>. <i>coli</i> RNase H (ecRNH) fragments have very similar stabilities, suggesting that the presence of additional residues in the I<sub>core+1</sub> fragment does not affect the folding or structure as compared to I<sub>core</sub>. NMR experiments provide additional evidence that only the I<sub>core</sub> intermediate is populated by ecRNH. This is one of the biggest differences that has been observed between the energy landscapes of these two proteins. Additionally, we used a FRET experiment in the background of full-length ttRNH to specifically monitor the formation of the I<sub>core+1 </sub>intermediate. We determine that the ttRNH I<sub>core+1 </sub>intermediate is likely the intermediate populated prior to the rate-limiting barrier to global folding, in contrast to <i>E</i>. <i>coli</i> RNase H for which I<sub>core </sub>is the folding intermediate. This result provides new insight into the nature of the rate-limiting barrier for the folding of RNase H.Towards the Systematic Mapping and Engineering of the Protein Prenylation Machinery in <i>Saccharomyces cerevisiae</i>Viktor SteinMarta H. KubalaJason SteenSean M. GrimmondKirill Alexandrov10.1371/journal.pone.01207162015-03-13T14:00:00Z2015-03-13T14:00:00Z<p>by Viktor Stein, Marta H. Kubala, Jason Steen, Sean M. Grimmond, Kirill Alexandrov</p>
Protein prenylation is a widespread and highly conserved eukaryotic post-translational modification that endows proteins with the ability to reversibly attach to intracellular membranes. The dynamic interaction of prenylated proteins with intracellular membranes is essential for their signalling functions and is frequently deregulated in disease processes such as cancer. As a result, protein prenylation has been pharmacologically targeted by numerous drug discovery programs, albeit with limited success. To a large extent, this can be attributed to an insufficient understanding of the interplay of different protein prenyltransferases and the combinatorial diversity of the prenylatable sequence space. Here, we report a high-throughput, growth-based genetic selection assay in <i>Saccharomyces cerevisiae</i> based on the Ras Recruitment System which, for the first time, has allowed us to create a comprehensive map of prenylatable protein sequences in <i>S</i>. <i>cerevisiae</i>. We demonstrate that potential prenylatable space is sparsely (6.2%) occupied leaving room for creation of synthetic orthogonal prenylatable sequences. To experimentally demonstrate that, we used the developed platform to engineer mutant farnesyltransferases that efficiently prenylate substrate motives that are not recognised by endogenous protein prenyltransferases. These uncoupled mutants can now be used as starting points for the systematic engineering of the eukaryotic protein prenylation machinery.Hyperphosphorylation of Intrinsically Disordered Tau Protein Induces an Amyloidogenic Shift in Its Conformational EnsembleShaolong ZhuAgnesa ShalaAlexandr BezginovAdnan SljokaGerald AudetteDerek J. Wilson10.1371/journal.pone.01204162015-03-13T14:00:00Z2015-03-13T14:00:00Z<p>by Shaolong Zhu, Agnesa Shala, Alexandr Bezginov, Adnan Sljoka, Gerald Audette, Derek J. Wilson</p>
Tau is an intrinsically disordered protein (IDP) whose primary physiological role is to stabilize microtubules in neuronal axons at all stages of development. In Alzheimer's and other tauopathies, tau forms intracellular insoluble amyloid aggregates known as neurofibrillary tangles, a process that appears in many cases to be preceded by hyperphosphorylation of tau monomers. Understanding the shift in conformational bias induced by hyperphosphorylation is key to elucidating the structural factors that drive tau pathology, however, as an IDP, tau is not amenable to conventional structural characterization. In this work, we employ a straightforward technique based on Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) and Hydrogen/Deuterium Exchange (HDX) to provide a detailed picture of residual structure in tau, and the shifts in conformational bias induced by hyperphosphorylation. By comparing the native and hyperphosphorylated ensembles, we are able to define specific conformational biases that can easily be rationalized as enhancing amyloidogenic propensity. Representative structures for the native and hyperphosphorylated tau ensembles were generated by refinement of a broad sample of conformations generated by low-computational complexity modeling, based on agreement with the TRESI-HDX profiles.