Alumni Dissertations

 

Alumni Dissertations

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  • A Role of Yeast Adhesin Amyloids in Force-Dependent Adhesion and Biofilm Formation

    Author:
    Cho Chan
    Year of Dissertation:
    2014
    Program:
    Biochemistry
    Advisor:
    Peter lipke
    Abstract:

    Candida albicans adhesins have amyloid-forming sequences (Ramsook et al. 2010, Otoo et al. 2008). Similarly, Tango and Waltz predicted that amyloid-forming sequences are also present in Saccharomyces cerevisiae flocculins, Flo1p and Flo11p. The cell surface of Flo1p- and Flo11p-expressing cells have ordered domains that are thioflavin T fluorescent and Congo red birefringent, two hallmarks of amyloids. Flo1p and Flo11p amyloids were important for activities of the flocculins including cell-to-cell adhesion, cell-to-substrate adhesion, and agar invasion. In addition, amyloid-perturbing dyes thioflavin S and Congo red inhibited aggregation, biofilm formation and agar invasion. Force-induced formation and propagation of adhesion nanodomains in Als5p-expressing cells were demonstrated with single-molecule atomic force microscopy (Alsteens et al. 2010). Because amyloid formation can be triggered by force, we investigated whether laminar flow and mechanical stress could activate amyloid formation and therefore increase adhesion. Shearing S. cerevisiae cells expressing Als5p or C. albicans at 0.8 dyne/cm2 increased quantity and strength of cell-to-surface and cell-to-cell binding, compared to 0.02 dyne/cm2. Mechanical stress from vortex-mixing at 2500 rpm also increased the aggregation of S. cerevisiae cells expressing Als5p or C. albicans. Similarly, cells expressing Flo1p and Flo11p also showed shear-and mechanical stress-dependent binding, and biofilm formation. I report here for the first time that catch bonding behavior in yeast cells was mediated by amyloid formation. Amyloids mediate both sensing and response in the presence of force. Adhesin-expressing cells binding to surfaces under shear stress were less likely to be washed off from the substrate than cells that were not stressed. This is characteristic of catch bonding. Catch bonding behavior was accompanied by the formation of amyloid nanodomains through the clustering at homotypic amyloid sequences. Thus, these nanodomains increased binding avidity of the adhesin-expressing cells to other cells (flocculation and aggregation assays) and to substrate surfaces. Furthermore we have devised ways of quantifying forces needed to activate aggregation, cell adhesion, and amyloids on the surface of yeast cells. Two different types of force, mechanical stress from vortex-mixing and shear stress from laminar flow increased adhesion and biofilm formation. Additionally, we quantified amyloid formation in live whole cell yeast suspensions in response to force. Fluorescent confocal microscopy and flow cytometry were used to quantify surface amyloids. Force-activated cells had punctate nanodomains with increased thioflavin T staining. Collectively, the assays can now be used to quantify amyloids in other fungal adhesins. These results demonstrate that 1. there are functional amyloids present in fungal adhesins Flo1p and Flo11p from S. cerevisiae, 2. amyloid formation mediates adhesion, agar invasion and biofilm, 3. amyloid nanodomains mediate force-sensitive catch-bonding, and 4. force-sensitive amyloid formation on the yeast cell wall surface can be quantified.

  • STRUCTURAL AND BIOCHEMICAL CHARACTERIZATION OF RHOMBOID INTRAMEMBRANE PROTEASES

    Author:
    Jose Chavez
    Year of Dissertation:
    2011
    Program:
    Biochemistry
    Advisor:
    Iban Ubarretxena
    Abstract:

    Intramembrane proteases are involved in multiple biological processes including cell growth and development, and apoptosis. There is no conservancy between hydrosoluble and membrane proteases. However, the catalytic residues and surrounding amino acids are absolutely conserved, suggesting that they both protein families share catalytic mechanisms but with two remarkable differences. (1) The ability of intramembrane proteases to cleave their substrates in the hydrophobic interior of the lipid bilayer, and (2) to do so in regions where the substrate displays a-helical conformation. D. melanogaster rhomboid-1 cleaves within the transmembrane domain region of epidermic growth factor receptor (EGFR) ligands Gurken, Keren and Spitz, resulting in their extracellular export. We designed substrate chimeras in which the transmembrane and cytoplasmic regions of Gurken, Keren and Spitz were preserved, while their EGFR ligand ectodomain was replaced by maltose binding protein. In vitro activity assays in detergent using purified components showed that rhomboid-1, H. sapiens RHBDL2, P. aeruginosa PA3086 and E. coli GlpG display comparable activity against these substrate chimeras. Mass spectrometry analysis of the N-terminal reaction product identified a single cleavage site after Ala138 for the Spitz chimeras, after Ala122 for the Keren chimeras, and after Ala245 for the Gurken chimeras that was identical for all rhomboids tested, suggesting a conservation of proteolytic profiles among prokaryotic and eukaryotic rhomboids. The identified cleavage site was located towards the N-terminal end of the transmembrane domain of each substrate. Positions that were sensitive to alanine scanning were further studied by introducing additional mutations to show that aside of ala in position P1, amino acids with low-helical propensities are necessary in the positions P2 and P1'. Finally, a bulky hydrophobic residue with a high helical propensity is important in P2' position to control the location of cleavage. We also carried out structural work and solved the N-terminal domain of Rhomboid and showed it displays high-affinity for membranes. Our work is put in a more general context by comparison with other intramembrane proteases and future work to unravel the mechanism of substrate binding and unwinding is also discussed.

  • STRUCTURAL AND BIOCHEMICAL CHARACTERIZATION OF RHOMBOID INTRAMEMBRANE PROTEASES

    Author:
    Jose Chavez
    Year of Dissertation:
    2011
    Program:
    Biochemistry
    Advisor:
    Iban Ubarretxena
    Abstract:

    Intramembrane proteases are involved in multiple biological processes including cell growth and development, and apoptosis. There is no conservancy between hydrosoluble and membrane proteases. However, the catalytic residues and surrounding amino acids are absolutely conserved, suggesting that they both protein families share catalytic mechanisms but with two remarkable differences. (1) The ability of intramembrane proteases to cleave their substrates in the hydrophobic interior of the lipid bilayer, and (2) to do so in regions where the substrate displays a-helical conformation. D. melanogaster rhomboid-1 cleaves within the transmembrane domain region of epidermic growth factor receptor (EGFR) ligands Gurken, Keren and Spitz, resulting in their extracellular export. We designed substrate chimeras in which the transmembrane and cytoplasmic regions of Gurken, Keren and Spitz were preserved, while their EGFR ligand ectodomain was replaced by maltose binding protein. In vitro activity assays in detergent using purified components showed that rhomboid-1, H. sapiens RHBDL2, P. aeruginosa PA3086 and E. coli GlpG display comparable activity against these substrate chimeras. Mass spectrometry analysis of the N-terminal reaction product identified a single cleavage site after Ala138 for the Spitz chimeras, after Ala122 for the Keren chimeras, and after Ala245 for the Gurken chimeras that was identical for all rhomboids tested, suggesting a conservation of proteolytic profiles among prokaryotic and eukaryotic rhomboids. The identified cleavage site was located towards the N-terminal end of the transmembrane domain of each substrate. Positions that were sensitive to alanine scanning were further studied by introducing additional mutations to show that aside of ala in position P1, amino acids with low-helical propensities are necessary in the positions P2 and P1'. Finally, a bulky hydrophobic residue with a high helical propensity is important in P2' position to control the location of cleavage. We also carried out structural work and solved the N-terminal domain of Rhomboid and showed it displays high-affinity for membranes. Our work is put in a more general context by comparison with other intramembrane proteases and future work to unravel the mechanism of substrate binding and unwinding is also discussed.

  • Protein Kinase C Substrates That Drive Motility of Cancer Cells

    Author:
    XIANGYU CHEN
    Year of Dissertation:
    2010
    Program:
    Biochemistry
    Advisor:
    Susan Rotenberg
    Abstract:

    As the intracellular receptor of tumor promoting phorbol esters, protein kinase C (PKC) is functionally linked to carcinogenesis and metastasis. Therefore, it is crucial to identify substrates of PKC in order to understand the mechanisms by which these substrate proteins participate in cancer-related phenotypes such as motile behavior. The work to be described consists of two projects: 1) new PKC substrates that contribute to the motility phenotype of human breast cells, and 2) the role of a known PKC substrate, MARCKS (Myristoylated Alanine-Rich C-Kinase Substrate) in the motility pathway of mouse melanoma cells. To identify direct substrates, a chemical-genetic approach was used to engineer the ATP binding site of PKC-α, δ and ζ to enable them to bind an unnatural ATP analogue. Consequently, phosphorylation is attributed exclusively to the mutant enzyme, and the phosphorylated protein bands are potential substrates. Following expression in human breast cells (MCF-10A), co-immunoprecipitation of the mutant enzyme bound with high affinity protein substrates was carried out. Following addition of an ATP analogue, a number of phosphorylated proteins were produced and subsequently analyzed by mass spectrometry. PKC-α and δ traceable kinases had very similar phosphorylation patterns, whereas the profile for PKC-ζ was distinct. Several potential protein substrates involved in cytoskeletal structure were identified for PKC-α and PKC-δ, including proteins that bind small GTP-binding proteins (Rho Kinase-1, Cdc42ep4, Rho/cdc42/Rac activating protein-1), as well as proteins that had been previously documented as PKC substrates (IQGAP, VASP). In a second project, the role of MARCKS was investigated in the context of mouse melanoma cell motility. As a protein that is known to crosslink actin bundles to the plasma membrane, this PKC substrate has been linked with cell migration and adhesion. Upon phosphorylation by PKC, MARCKS is released into the cytoplasm, thereby promoting actin rearrangement. Weakly metastatic B16 F1 cells do not express detectable phospho-MARCKS. However, F1 cells that are engineered to express a pseudo-phosphorylated mutant of MARCKS exhibit elevated motility. When F1 cells are treated with okadaic acid (OA), an inhibitor of protein phosphatases, increases in both phospho-MARCKS and motility are observed. OA-induced motility can be substantially eliminated when cells are pre-treated with a shRNA reagent to knock down MARCKS expression, or when expressing a phosphorylation-resistant mutant of MARCKS. Furthermore, a phosphorylation-resistant MARCKS mutant that was made exclusively cytoplasmic due to lack of a myristoyl group was observed to inhibit motility of OA-treated F1 cells as well as motility of highly metastatic melanoma cells (mouse F10 and human A375 cells). These findings imply that MARCKS promotes motility through cytoplasmic interactions involving its phosphorylated effector domain. It is concluded that phospho-MARCKS has a previously undocumented role in the cytoplasm that promotes motile behavior and possibly the metastatic potential of many different cancer cells. Overall, this work describes substrates of PKC that transmit the motility signal in cancer cells, and suggests novel targets for the development of anti-metastasis agents.

  • Protein Kinase C Substrates That Drive Motility of Cancer Cells

    Author:
    XIANGYU CHEN
    Year of Dissertation:
    2010
    Program:
    Biochemistry
    Advisor:
    Susan Rotenberg
    Abstract:

    As the intracellular receptor of tumor promoting phorbol esters, protein kinase C (PKC) is functionally linked to carcinogenesis and metastasis. Therefore, it is crucial to identify substrates of PKC in order to understand the mechanisms by which these substrate proteins participate in cancer-related phenotypes such as motile behavior. The work to be described consists of two projects: 1) new PKC substrates that contribute to the motility phenotype of human breast cells, and 2) the role of a known PKC substrate, MARCKS (Myristoylated Alanine-Rich C-Kinase Substrate) in the motility pathway of mouse melanoma cells. To identify direct substrates, a chemical-genetic approach was used to engineer the ATP binding site of PKC-α, δ and ζ to enable them to bind an unnatural ATP analogue. Consequently, phosphorylation is attributed exclusively to the mutant enzyme, and the phosphorylated protein bands are potential substrates. Following expression in human breast cells (MCF-10A), co-immunoprecipitation of the mutant enzyme bound with high affinity protein substrates was carried out. Following addition of an ATP analogue, a number of phosphorylated proteins were produced and subsequently analyzed by mass spectrometry. PKC-α and δ traceable kinases had very similar phosphorylation patterns, whereas the profile for PKC-ζ was distinct. Several potential protein substrates involved in cytoskeletal structure were identified for PKC-α and PKC-δ, including proteins that bind small GTP-binding proteins (Rho Kinase-1, Cdc42ep4, Rho/cdc42/Rac activating protein-1), as well as proteins that had been previously documented as PKC substrates (IQGAP, VASP). In a second project, the role of MARCKS was investigated in the context of mouse melanoma cell motility. As a protein that is known to crosslink actin bundles to the plasma membrane, this PKC substrate has been linked with cell migration and adhesion. Upon phosphorylation by PKC, MARCKS is released into the cytoplasm, thereby promoting actin rearrangement. Weakly metastatic B16 F1 cells do not express detectable phospho-MARCKS. However, F1 cells that are engineered to express a pseudo-phosphorylated mutant of MARCKS exhibit elevated motility. When F1 cells are treated with okadaic acid (OA), an inhibitor of protein phosphatases, increases in both phospho-MARCKS and motility are observed. OA-induced motility can be substantially eliminated when cells are pre-treated with a shRNA reagent to knock down MARCKS expression, or when expressing a phosphorylation-resistant mutant of MARCKS. Furthermore, a phosphorylation-resistant MARCKS mutant that was made exclusively cytoplasmic due to lack of a myristoyl group was observed to inhibit motility of OA-treated F1 cells as well as motility of highly metastatic melanoma cells (mouse F10 and human A375 cells). These findings imply that MARCKS promotes motility through cytoplasmic interactions involving its phosphorylated effector domain. It is concluded that phospho-MARCKS has a previously undocumented role in the cytoplasm that promotes motile behavior and possibly the metastatic potential of many different cancer cells. Overall, this work describes substrates of PKC that transmit the motility signal in cancer cells, and suggests novel targets for the development of anti-metastasis agents.

  • Structure Determination of a Double Transmembrane Fragment of the G protein-coupled Receptor Ste2p in Membrane Mimetic Environments

    Author:
    Leah Cohen
    Year of Dissertation:
    2010
    Program:
    Biochemistry
    Advisor:
    Fred Naider
    Abstract:

    G-protein coupled receptors (GPCRs) are relevant in cellular signal transduction pathways and are targets for disease therapeutics. Since the sequencing of the human genome, there have been close to 1000 GPCRs predicted and many have been characterized by biological and biochemical analysis. Though these integral membrane proteins (IMPs) have little sequence similarity, they show strong putative structural similarities. All GPCRs contain an N-terminal extracellular domain (NT), 7 transmembrane helical regions (TM) connected by intra- and extracellular loops (IL and EL, respectively), and a C-terminal intracellular tail (CT). The extracellular domains are thought to play a role in ligand-receptor interactions and together with the TM domains form the ligand binding site for many GPCRs. The number of structures in the protein structural database is increasing exponentially, but there are only a few high-resolution GPCR structures, those of rhodopsin, the β-adrenergic receptors and the adenosine A2A receptor. GPCRs are difficult to crystallize and the protein:detergent complex size can be restrictive for solution NMR. As is true of GPCRs in general, limited structural information is available for Ste2p, a yeast GPCR which recognizes the α-factor tridecapeptide mating pheromone. Many groups, including the Naider lab, have been working with smaller fragments of GPCRs in an attempt to elucidate the structure of individual regions by either crystallization or NMR and to combine these to achieve a better picture of GPCR structure. The use of fragments of GPCRs to study the structure of these large membrane receptors remains controversial. I have used Ste2p as a paradigm to answer the following questions: 1) Can GPCR fragments fold into a tertiary structure without the context of the full protein? and 2) Do organic:aqueous solvents such as trifluoroethanol(TFE):water result in GPCR fragment structures similar to those found in detergents and lipids? I have cloned and expressed two 2TM regions of Ste2p, G31-T110 (TM1-TM2) and R231-S339 (TM6-TM7-CT40). The constructs were chosen based on the biological relevance of the domain and a buried surface area analysis that was performed using a rhodopsin-templated model of Ste2p and the NACCESS software package. Expression in Escherichia coli in minimal media and CNBr cleavage conditions for these 2TM fusion proteins were optimized and the fusion protein was labeled with [15N], [15N,13C], [15N,13C,2H] and with selectively [15N]-labeled amino acids to aid in NMR assignments. Purification of isotopically labeled peptides was performed using RP-HPLC with a Zorbax C3 column and an acetonitrile:water gradient containing isopropanol and 0.1% TFA. Yields of Ste2p(G31-T110) were 6-20 mg per L culture. Far UV CD analysis indicated that Ste2p(G31-T110) formed a highly helical peptide in TFE:water and various micellar environments, whereas the helicity was reduced for Ste2p(R231-S339). Initial [15N,1H]-HSQC analysis showed that CD can be used as an NMR screening technique to determine conditions for high resolution NMR analysis. Ste2p(G31-T110) was used to determine a high-resolution structure of a 2TM GPCR fragment in TFE:water(+0.1% TFA) (1:1; v/v) by multidimensional NMR spectroscopy. The 80-residue polypeptide was folded into a helical hairpin, but the overall convergence of the structure bundle was poor with an RMSD of ~ 6.7 Å. Comparison to an NMR study of TM1-TM2 in LPPG micelles concluded that very different structures occurred in the two membrane mimetic media and it was observed that relative conformation of TM2 to TM1 in TFE:water was twisted in comparison to that in the LPPG micelle so that the interactions between helices were on opposite sides. Although Ste2p(G31-T110) folds into a tertiary structure in TFE:water (1:1; v/v) the structure is quite flexible compared to that in LPPG micelles.

  • Mechanism of Phospho-alpha-Tubulin-driven Motility in Human Breast Epithelial Cells

    Author:
    Shatarupa De
    Year of Dissertation:
    2014
    Program:
    Biochemistry
    Advisor:
    Susan Rotenberg
    Abstract:

    Protein kinase C (PKC), an enzyme important in signaling pathways that give rise to various cell phenotypes, has been closely associated with metastatic phenotypes of breast cancer. Tissues from patients of varying degrees of tumorgenicity have shown an elevated expression of the PKCα isoform as well as other diacylglycerol (DAG)-sensitive isoforms. Moreover, independent studies with highly invasive breast cell lines, such as MDA-MB-231 cells, have also shown an elevated level of PKCα. Therefore, PKCα has long been a therapeutic target for breast cancer. Among its known substrates (recently identified by our laboratory), α6-tubulin will, upon phosphorylation by PKC, enhance migration in breast epithelial cells. The mechanistic pathway as to how a phosphorylated state of α-tubulin can confer motility in breast epithelial cells was addressed in the present work. Different approaches were undertaken to dissect the role(s) played by phospho-α6-tubulin in enhancing cell migration: i) extensive live cell imaging to quantitate microtubule dynamics, ii) immunofluorescence of microtubules in fixed cells, iii) partitioning of phospho-α6-tubulin between particulate and soluble phases, and iv) pull-down assays to determine effects on the small GTPases. For these studies, two mutants of α6-tubulin namely, S165D (pseudo-phosphorylated) and S165N (phosphorylation-resistant), were used extensively. In addition, cells expressing wildtype (WT)-α6-tubulin were treated with either the PKC-activating DAG-lactone or the pan-PKC inhibitor bis-indoleylmaleimide (BIM). Experiments were conducted in non-motile MCF-10A cells that have a low expression level of PKC and therefore offer an ideal platform for studying PKC-mediated changes. Some experiments were also conducted in MDA-MB-231 metastatic breast epithelial cells to re-affirm the role of phospho-α-tubulin in motility and related phenotypes. Functionality of phospho-α-tubulin was also explored by examining its interaction with the microtubule cargo protein MARCKS (a PKC-substrate), and membrane proteins like IQGAP1 and Rac1 having known involvement in cell migration. Each α6-tubulin mutant was studied for its effects on the distinct morphological glandular structures that it produced in a 3D growth environment, in order to simulate an in vivo condition. This study is the first of its kind to establish a detailed mechanism of cell movement driven by the PKC substrate α-tubulin in human breast cells. The novel role of phospho-α-tubulin in promoting motility will establish it as an important predictive marker for metastatic breast cancer.

  • The microtubule associated protein tau renders breast cancer cells TNF-alpha resistant by inhibiting TNF-receptor signaling.

    Author:
    Shawon Debnath
    Year of Dissertation:
    2013
    Program:
    Biochemistry
    Advisor:
    Jimmie Fata
    Abstract:

    The pro-inflammatory cytokine Tumor Necrosis Factor &alpha is often found in elevated concentration within the microenvironment of breast tumors. A number of findings have now established that TNF&alpha can exert opposing effects on tumor cells - acting either as an anti-cancer agent or as a promoter of tumor progression. To date, mechanisms underlying these divergent outcomes have not been elucidated. Here, we demonstrate that tau, classically considered as a microtubule-associated protein, plays a key role to determine whether cancer cells respond negatively (apoptosis) or positively (proliferation) to TNF&alpha exposure. Using RNAi knockdown experiments we show that up-regulation of tau protein in breast cancer cells is necessary for the acquisition of resistance to TNF&alpha mediated cytotoxicity. In contrast, an analysis of generated stable cell lines overexpressing full-length tau indicates that tau can inhibit TNF&alpha induced caspase activation and NFκB nuclear translocation. Site-directed mutagenesis has revealed that the N-terminal portion of tau, which does not bind to tubulin, is sufficient for this inhibition of TNF&alpha signaling. Finally, mechanistic studies have uncovered that tau inhibits TNF-receptor trimerization and receptor clustering thereby blocking subsequent signaling. Taken together, we conclude that acquisition of TNF&alpha resistance requires a previously undescribed mechanism involving up-regulation of tau, which in turn inhibits receptor trimerization and thus attenuates TNF&alpha downstream signaling in tumor cells.

  • Mechanisms of regulation of mRNA 3' processing by p53 pathway

    Author:
    Emral Devany
    Year of Dissertation:
    2014
    Program:
    Biochemistry
    Advisor:
    Frida Kleiman
    Abstract:

    Mechanisms of regulation of mRNA 3' processing by p53 pathway by Emral Cakmak Devany Adviser: Professor Frida Esther Kleiman Although the p53 network has been intensively studied, genetic analyses long hinted at the existence of components that remained elusive. This dissertation focuses on the study of the regulation of mRNA 3' processing during DNA damage response (DDR) by the p53 pathway and the regulation of p53 expression by the mRNA 3' processing machinery. The results in this dissertation revealed new roles of tumor suppressor p53 in mRNA 3' processing. In Chapter II, I showed that p53 inhibits the cleavage step of polyadenylation reaction and that cells with different levels of p53 expression have different mRNA processing profiles. As part of the same response to DNA damage, my results indicate that p53 also activates PARN-dependent deadenylation in the nucleus (Chapter III). In Chapter IV, I demonstrated that p53 mRNA is one of the biological targets of nuclear PARN under non-stress conditions. Extending these studies, in Chapter V, I established that both AU-rich element (ARE) and miR-125b binding site are important for the binding of PARN to the p53 mRNA and activation of p53 pathway. Together these results show a feedback loop between PARN deadenylase and one of its targets, the tumor suppressor p53: While PARN keeps p53 levels low by destabilizing p53 mRNA through ARE- and microRNA-binding sites in non-stress conditions; the increase in p53 levels after UV treatment results in the activation of PARN deadenylase in a transcription-independent manner. As the levels of p53 expression levels increase after DNA damage, the PARN-mediated down-regulation of p53 mRNA should be reverted during the progression of DDR. In Chapter VI, I found that under DNA damaging conditions HuR, a ubiquitously expressed ARE-binding protein, can compete for binding to the p53 3'UTR with both PARN and Ago-2, resulting in the release of PARN and Ago-2 from p53 mRNA and the increase of p53 expression levels. Finally in Chapter VII, I analyzed the usage of alternative polyadenylation signals (APA) during DDR. My results indicate that increase in intronic-polyadenylated isoforms of genes involved in DDR occurred after UV treatment, indicating that APA might represent another potential mechanism of controlling gene expression during the response to DNA damage. Together this dissertation provides new insights into p53 function and the mechanisms behind the regulation of mRNA 3' end processing and hence gene expression in different cellular conditions.

  • Turnip Mosaic Virus Genome-Linked Protein (VPg) Inhibits Pokeweed Antiviral Protein (PAP)-Mediated Depurination of RNA

    Author:
    Artem Domashevskiy
    Year of Dissertation:
    2011
    Program:
    Biochemistry
    Advisor:
    Dixie Goss
    Abstract:

    Pokeweed antiviral protein (PAP) from Phytolacca americana is a ribosome inactivating protein (RIP) and is an RNA N-glycosidase that removes specific purine residues from the sarcin/ricin (S/R) loop of large rRNA, arresting protein synthesis at the translocation step. PAP is a cap-binding protein, and it was suggested that it inhibits translation of RNA by binding to the 5' m7G cap structure of eukaryotic mRNA, and depurinating the mRNA at sites downstream of the cap structure. PAP is a potent antiviral agent against many plant, animal, and human viruses. Depurination of capped viral RNA may be the primary mechanism for PAP's antiviral activity. However, the above mechanism does not clarify the inhibitory effect of PAP on the replication of uncapped viruses. To elucidate the mechanism of RNA depurination, and to understand how PAP recognizes and targets various RNAs, the interactions between PAP and Turnip mosaic virus (TuMV) genome linked protein (VPg) were investigated. VPg is important in the initiation of protein synthesis, functioning as a cap analog. VPg stimulates the in vitro translation of uncapped IRES-containing RNA and inhibits capped RNA translation in wheat germ extracts. In this work, fluorescence spectroscopy and HPLC techniques were used to quantitatively describe PAP-VPg interactions. PAP interacts strongly with VPg, thus the effect of VPg on the PAP catalyzed depurination of several different RNA molecules was determined to investigate whether VPg binding to PAP influences selectivity of depurination. PAP binds to and depurinates both m7GpppG-capped and uncapped S/R oligo nucleotide and TEV RNAs, supporting previous conclusions that the cap structure is not the only determinant for PAP depurination of RNA. VPg decreases depurination of the above capped and uncapped RNAs and competes with TEV RNA for PAP binding. VPg may confer an evolutionary advantage by suppressing one of the defense mechanisms of the plant. Depurination inhibition of PAP by VPg also suggests the possible use of this protein against cytotoxic activity of RIPs and inhibition of their biological potency.