Avidin Beads Research Articles

A Chemoproteomics Approach to Determine the Mechanism of Testicular Toxicity for the Bruton’s Tyrosine Kinase Inhibitor CC-292

During drug development, potential safety issues can occur at any time. Understanding the cause of a toxicity can help with deciding on how to advance the drug development program. Chemoproteomics provides a way to help understand the cause of a toxicity wherein the affected tissue is accessible and can be probed with a covalently binding compound that is analogous to the offending drug. In this case, N-(3-(5-fluoro-2-(4-(2-methoxyethoxy)phenylamino)pyrimidin-4-ylamino)phenyl)acrylamide (CC-292), a covalently binding Bruton's tyrosine kinase inhibitor, had produced testicular toxicity in rodents. Experiments were conducted using a CC-292 analog that could be chemically modified with biotin to probe rodent testes homogenates for potential binding sites that were subsequently recovered with avidin beads. These biotin-tagged proteins undergo trypsin digest on the avidin beads to yield peptides that are identified using mass spectrometry. Two proteins were identified from the testicular homogenates of both rats and mice, namely retinal dehydrogenase 1 (ALDH1A1) and retinal dehydrogenase 2 (ALDH1A2). Literature confirmed a link between inhibition of these enzymes and testicular toxicity. Subsequently, molecular modeling was used to demonstrate that CC-292 can be docked into both the nicotinamide adenine dinucleotide and retinal binding pockets of the analogous human ALDH1A2 enzyme. These data suggest that the off-target binding site for CC-292 on retinal dehydrogenase enzymes may provide a mechanistic explanation to the testicular toxicity observed in rodents and that there may be a potential concern for human male fertility. SIGNIFICANCE STATEMENT: Biotinylated covalently binding drug analogues are used to enrich bound proteins from tissue homogenates wherein toxicity was observed in rodents. Bound proteins were subsequently identified by mass spectroscopy. Competition of the analog binding with the parent inhibitor itself and three-dimensional molecular modeling were used to establish a likely link between the off-targets of CC-292, ALDH1A1, and ALDH1A2 with potential testicular toxicity.

Optimization of Cell Free Protein Synthesis: Development of a Fusion Protein Fitness Strategy

Our undergraduate researchers work in the field of metabolic engineering to harness metabolic pathways for the production of useful metabolites with applications in the biofuel, pharmaceutical, or industrial chemical industries. We published a metabolic engineering approach called programmed evolution that introduces variation in regulatory elements for the expression of enzymes that control orthogonal metabolic pathways. Programmed evolution employs a riboswitch‐based fitness module to transduce the production of a desired metabolite into antibiotic resistance. We validated programmed evolution by optimizing the conversion of caffeine to theophylline by caffeine demethylase, but we recognized several limitations to the widespread use of our approach, including crosstalk between orthogonal and native bacterial metabolism, potential toxicity of metabolites, and poor representation of large variation spaces. To avoid these limitations, we are developing an in vitro approach to programmed evolution. For cell‐free protein synthesis (CFPS), we found supercoiled plasmid was a much better template than linear PCR amplicon. We designed, constructed, and have begun testing a novel fitness module that responds to the production of theophylline produced by the action of caffeine demethylase produced by CFPS. The fitness module encodes a fusion protein composed of three functional units. The amino portion contains a Gal4 DNA binding domain that will target binding sites on a library of DNA regulatory elements for the expression of caffeine demethylase. The carboxyl terminus includes streptavidin which will bind to avidin beads for physical separation of selected DNA regulatory elements. Sandwiched in between these two domains is GFP, which serves as a biosensor. Using this in vitro selection strategy, we plan to pursue the optimization of CFPS for the discovery of efficient metabolic pathways of interest.Support or Funding InformationNSF RUI grant MCB‐1613203 to Davidson College and MCB‐1613281 to Missouri Western State University, Davidson College Martin Genomics ProgramThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Investigation of A Mechanical Application of The Avidin Beads Bonded Bacirus substilis as Living Micromachines

Investigation of A Mechanical Application of The Avidin Beads Bonded Bacirus substilis as Living Micromachines

Utilization of Polymer Based Protein Engineering and ATRP to Modulate Substrate Size Specificity of Avidin

Avidin and biotin interact very strongly and are commonly used to tag and purify proteins or peptides for proteomic analysis. Using a pH dependent reversible biotin tag that targets primary amines, all the proteins can be tagged at a slightly basic pH. After labeling proteins with the biotin tag, any excess unreacted biotin left over in solution must be dialyzed away to prevent competition for binding spots on the Avidin beads. Tagged proteins can then be bound to a solid surface Avidin bead and contaminants washed away. Proteins can then be released from the beads in their original unmodified state by lowering the pH. While this workflow has been shown to be very useful for purifying proteomes, the requirement for dialysis after the initial tagging step makes this workflow laborious and difficult to automate. This step is time consuming, can result in protein loss or precipitation, and makes automation impossible. In this study we seek to utilize polymer based protein engineering to circumvent this issue.Atom transfer radical polymerization (ATRP) has been utilized in recent years to specifically modify and fine tune the behavior of proteins. By adding polymers of a specific composition and length in targeted areas of a protein, the behavior of a protein can be modified. In this study, we utilized ATRP to grow a dense polymer network off the surface of Avidin to act as a molecular sieve; The large polymers allow small molecules of biotin to bind to the binding site, while larger biotinylated proteins are excluded. The resulting “Caged Avidin” can then be used in the proteome purification workflow in the place of dialysis by binding and sequestering any unreacted free biotin, allowing the entire labeling, binding, washing, and eluting workflow to take place in a single tube with no bias and no sample loss. This can revolutionize sample preparation in biomarker and drug discovery by allowing for automation, and thus improved yield and reliability, of sample preparation.Support or Funding InformationThis work was supported in part by the Center for Polymer Based Protein Engineering at Carnegie Mellon UniversityThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

ProC-TEL: Profiling of Protein C-Termini by Enzymatic Labeling.

Proteins are frequently processed by proteases in cell signaling pathways to perform their biological functions in response to environmental stimuli. Identification of the exact cleavage sites provides necessary information for the study of their biological functions. Although proteomic approaches for profiling of protein N-termini have been developed extensively in the past few years, the N-terminal profiling strategy has its inherent disadvantages. Therefore, C-terminal profiling approaches might be a complementary approach for the identification of protein cleavages although it has similar shortcomings as N-terminal profiling methods. In this protocol, we describe an approach, termed ProC-TEL: Profiling of Protein C-Termini by Enzymatic Labeling, for affinity labeling of protein C-termini for a protein or proteome. This method uses the transpeptidase activity of carboxypeptidase Y to label protein C-termini with an affinity biotin tag for subsequent isolation with avidin beads and identification by mass spectrometer. It is complementary to the N-terminal profiling approaches and can be used to identify proteolytic cleavages for a specific protease or in cell signaling events, such as apoptosis.

Drug Target Identification Using an iTRAQ-Based Quantitative Chemical Proteomics Approach-Based on a Target Profiling Study of Andrographolide.

Identifying the cellular binding targets of drugs and other bioactive small molecules is a crucial step for understanding their molecular mechanisms of action as well as potential off-target effects. The field of chemical proteomics is an emerging discipline in chemical biology using synthetic chemistry and high-throughput detection techniques to study small molecule-protein interactions. In this chapter, we describe a quantitative chemical proteomics protocol combining bioorthogonal click chemistry and quantitation by isobaric tags for relative and absolute quantification (iTRAQ) to identify the specific binding targets of drugs and bioactive small molecules such as natural products. A modified drug probe with a click chemistry-enabling addition is synthesized and used in live cell treatments where it undergoes covalent interactions with its cognate cellular targets. The probes are then ligated to biotin through click chemistry and enriched with avidin beads, followed by iTRAQ labeling and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis for protein identification and relative quantitation discriminating specific targets from nonspecific binding proteins. The presented protocol has been used to successfully profile prominent drugs and natural products including andrographolide, aspirin, curcumin, etc., and can be a powerful tool to study the molecular mechanisms of bioactive small molecules.

Proteomic Profiling of De Novo Protein Synthesis in Starvation-Induced Autophagy Using Bioorthogonal Noncanonical Amino Acid Tagging.

Autophagy is an intracellular degradation process activated by stress factors such as nutrient starvation to maintain cellular homeostasis. There is emerging evidence demonstrating that de novo protein synthesis is involved in the autophagic process. However, up-to-date characterizing of these de novo proteins is technically difficult. In this chapter, we describe a novel method to identify newly synthesized proteins during starvation-mediated autophagy by bioorthogonal noncanonical amino acid tagging (BONCAT), in conjunction with isobaric tagging for relative and absolute quantification (iTRAQ)-based quantitative proteomics. l-azidohomoalanine (AHA) is an analog of methionine, and it can be readily incorporated into the newly synthesized proteins. The AHA-containing proteins can be enriched with avidin beads after a "click" reaction between alkyne-bearing biotin and the azide moiety of AHA. The enriched proteins are then subjected to iTRAQ™ labeling for protein identification and quantification using liquid chromatography-tandem mass spectrometry (LC-MS/MS). By using this technique, we have successfully profiled more than 700 proteins that are synthesized during starvation-induced autophagy. We believe that this approach is effective in identification of newly synthesized proteins in the process of autophagy and provides useful insights to the molecular mechanisms and biological functions of autophagy.

Quantitative chemical proteomics profiling of de novo protein synthesis during starvation-mediated autophagy

ABSTRACTAutophagy is an intracellular degradation mechanism in response to nutrient starvation. Via autophagy, some nonessential cellular constituents are degraded in a lysosome-dependent manner to generate biomolecules that can be utilized for maintaining the metabolic homeostasis. Although it is known that under starvation the global protein synthesis is significantly reduced mainly due to suppression of MTOR (mechanistic target of rapamycin serine/threonine kinase), emerging evidence demonstrates that de novo protein synthesis is involved in the autophagic process. However, characterizing these de novo proteins has been an issue with current techniques. Here, we developed a novel method to identify newly synthesized proteins during starvation-mediated autophagy by combining bio-orthogonal noncanonical amino acid tagging (BONCAT) and isobaric tags for relative and absolute quantitation (iTRAQTM). Using bio-orthogonal metabolic tagging, L-azidohomoalanine (AHA) was incorporated into newly synthesized proteins which were then enriched with avidin beads after a click reaction between alkyne-bearing biotin and AHA's bio-orthogonal azide moiety. The enriched proteins were subjected to iTRAQ labeling for protein identification and quantification using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Via the above approach, we identified and quantified a total of 1176 proteins and among them 711 proteins were found to meet our defined criteria as de novo synthesized proteins during starvation-mediated autophagy. The characterized functional profiles of the 711 newly synthesized proteins by bioinformatics analysis suggest their roles in ensuring the prosurvival outcome of autophagy. Finally, we performed validation assays for some selected proteins and found that knockdown of some genes has a significant impact on starvation-induced autophagy. Thus, we think that the BONCAT-iTRAQ approach is effective in the identification of newly synthesized proteins and provides useful insights to the molecular mechanisms and biological functions of autophagy.

Enhanced Sample Preparation for Proteomic Analysis Utilizing Reversible Biotinylation and Polymer Based Protein Engineering

Proteome sample preparation for gel‐based and mass spectrometry‐based studies has remained relatively unchanged for many years. The goal of these sample preparation schemes is to remove contaminants such as lipids, DNA, RNA, and carbohydrates from cell or tissue homogenates. The vast majority of current methods rely on protein precipitation followed by centrifugation and resolubilizing the proteins. Unfortunately none of these techniques provide an unbiased sampling of the tentire proteome. Regardless of the method (ammonium acetate precipitation, TCA/acetone precipitation, PEG precipitation, etc.), not all proteins will be precipitated and not all will be resolubilized. We have developed an unbiased method for proteome sample preparation based on protein capture, not precipitation. We recently showed that Biotin‐CDM can be used as a tool to reversibly tag the entire proteome for purification.1 Biotin‐CDM reacts with lysine residues present on the surface of proteins at elevated pH to covalently biotinylate proteins. Biotinylated proteins are bound to Avidin beads and then all non‐protein contaminants are washed away. The biotin tag is then reversed by lowering the pH to release the proteins from the Avidin beads, resulting in pur, unmodified, and unbiased proteome sample. Because an excess of Biotin‐CDM is required to tag all proteins, we utilized polymer based protein engineering to develope a novel method that separates free Biotin‐CDM from biotinylated proteins. Thus, we have developed a method to prepare proteome samples in an unbiased manner that could be used for any organism.

Asp1 from Schizosaccharomyces pombe binds a [2Fe-2S](2+) cluster which inhibits inositol pyrophosphate 1-phosphatase activity.

Iron-sulfur (Fe-S) clusters are widely distributed protein cofactors that are vital to cellular biochemistry and the maintenance of bioenergetic homeostasis, but to our knowledge, they have never been identified in any phosphatase. Here, we describe an iron-sulfur cluster in Asp1, a dual-function kinase/phosphatase that regulates cell morphogenesis in Schizosaccharomyces pombe. Full-length Asp1, and its phosphatase domain (Asp1(371-920)), were each heterologously expressed in Escherichia coli. The phosphatase activity is exquisitely specific: it hydrolyzes the 1-diphosphate from just two members of the inositol pyrophosphate (PP-InsP) signaling family, namely, 1-InsP7 and 1,5-InsP8. We demonstrate that Asp1 does not hydrolyze either InsP6, 2-InsP7, 3-InsP7, 4-InsP7, 5-InsP7, 6-InsP7, or 3,5-InsP8. We also recorded 1-phosphatase activity in a human homologue of Asp1, hPPIP5K1, which was heterologously expressed in Drosophila S3 cells with a biotinylated N-terminal tag, and then isolated from cell lysates with avidin beads. Purified, recombinant Asp1(371-920) contained iron and acid-labile sulfide, but the stoichiometry (0.8 atoms of each per protein molecule) indicates incomplete iron-sulfur cluster assembly. We reconstituted the Fe-S cluster in vitro under anaerobic conditions, which increased the stoichiometry to approximately 2 atoms of iron and acid-labile sulfide per Asp1 molecule. The presence of a [2Fe-2S](2+) cluster in Asp1(371-920) was demonstrated by UV-visible absorption, resonance Raman spectroscopy, and electron paramagnetic resonance spectroscopy. We determined that this [2Fe-2S](2+) cluster is unlikely to participate in redox chemistry, since it rapidly degraded upon reduction by dithionite. Biochemical and mutagenic studies demonstrated that the [2Fe-2S](2+) cluster substantially inhibits the phosphatase activity of Asp1, thereby increasing its net kinase activity.

Abstract 694: Structure-function analysis of RPL18A, a putative binding target of rigosertib

Abstract Rigosertib (ON 01910.Na; RGS) is a clinical stage anticancer agent that causes spindle abnormalities and mitotic arrest in neoplastic cells. The drug inhibits PI3K and PLK1 signaling pathways, down regulates cyclin D1 expression and induces apoptosis. Previously, we reported identification of RPL18A (L18A), a protein from the large ribosomal subunit, as a putative binding target of RGS [Proc. AACR 2014, #4595]. Knock-down of L18A with siRNA caused apoptosis in cancer cell lines. Role of L18A in the function of the ribosome is not known. Goal of this study was to conduct structure-function analysis of L18A and create deficiency mutant(s) for future experiments. Human L18A is predicted to adopt the following linear order of secondary structural elements - alpha helices (a) and beta strands (b) - when assembled into the ribosome - b1b2a1b3b4b5b6a2a3b7a4 (from www.rcsb.org/pdb/protein/Q02543). The predicted 3D structure of L18A suggests overall bilobal architecture with a flanking C-terminal tail. Packed in tandem, each lobe contains one alpha helix surrounded by a beta sheet formed by 4 (N-lobe) or 3 (C-lobe) beta strands. To map the potential binding site for RGS, we engineered multiple mutants of L18A including truncations at its N and C termini. Our approach involved successive removal of the structural elements and a few substitutions with alanine. Mutant proteins were expressed in E.coli as GST fusions, and derivative cell lysates were tested in pull-down assay with biotin-conjugated RGS (RGSbio) and avidin beads. Pulled-down protein-drug complexes were analyzed by Western blot with anti-GST antibody. At the C-terminus, abolishing a4 helix (C - a4) preserved RGSbio-binding activity comparable to wild type (WT) L18A. Further C-truncations resulted in partial (C - a3b7a4) or complete loss of activity (C - a2→a4; C - b6→a4 and C - b5→a4; latter represents N-lobe while missing entire C-lobe). Unlike N-lobe, independently expressed C-lobe had specific binding activity, albeit half that of WT. At the N-terminus, stepwise truncation of amino acids 6 through 12 in the first beta strand (b1) resulted in gradual reduction of specific binding activity. To verify that this was not due to structure misfolding we tested mutants with single Ala substitutions in b1 and obtained similar results. Such mutants will serve as RGS-non-binding controls in future studies. In the model, L18A packs in the ribosome in a way that its surface at the side of two alpha helices of both lobes is engaged in protein-RNA interactions. However, the outer surface of the two cross-braced beta sheets in both lobes is completely exposed to solvent and interestingly, there is a cavity formed at the junction of two lobes that appears to be deep enough to accommodate one molecule of RGS. Taken together, our data suggest that rigosertib approaches RPL18A at the exposed hinge region between the two lobes and may thus perturb ribosomal function in cancer cells. Citation Format: Irina A. Oussenko, Yogesh K. Gupta, Rodrigo Vasquez-Del Carpio, M.V. Ramana-Reddy, Aneel K. Aggarwal, E. Premkumar Reddy, James F. Holland, Takao Ohnuma. Structure-function analysis of RPL18A, a putative binding target of rigosertib. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 694. doi:10.1158/1538-7445.AM2015-694

Reminiscences on my life with RNA: a self-indulgent perspective.

Reminiscences on my life with RNA: a self-indulgent perspective.

Abstract 4595: RPL18A as putative target of rigosertib

Abstract Rigosertib (ON 01910.Na) is clinical stage anti-mitotic cancer inhibitor that causes spindle abnormalities in neoplastic cells. Rigosertib inhibits PI3K and PLK1 signaling pathways, down regulates cyclin D1 expression and induces apoptosis. Previously we reported rigosertib led to hyperphosphorylation of RanGAP1•SUMO1, regulator of RanGTP/RanGDP cycle, and hypothesized phosphatase inhibition [Cancer Res 2011; 71; 4968-76]. Using biotin-conjugated rigosertib and avidin-conjugated agarose beads, we sought rigosertib molecular target from HeLa cell lysates by pull-down assay. Long-linker 1910-biotin showed an IC50 for cytotoxicity similar to unmodified rigosertib and efficient avidin binding. Asynchronously grown HeLa cell lysates were incubated with designated ligands and avidin beads. Bound lysate proteins were recovered from beads, resolved on SDS-PAGE and stained. Specific protein band of ∼20 kDa was detected only with long-linker 1910-biotin. Identification of proteins from the gel was performed by LC-ESI-MS/MS. Four ribosomal proteins possessed Mascot score above cut-off level with RPL18A, score of 367, and other proteins scored 153 or less. Specific dose-dependent binding of long-linker 1910-biotin to RPL18A was confirmed by Western blot analysis in replicate pull-down experiments using HeLa or MOLT-3 cell lysates. Recombinant RPL18A possessed specific reactivity to 1910-biotin in the pull-down assay. Knock-down of RPL18A with siRNA causes apoptosis in cancer cell lines [Sudo et al, Genomics 2010; 95; 210-6; and our data]. We propose that nucleocytoplasmic transport of RPL18A is impaired when hyperphosphorylated RanGAP1•SUMO1 fails to provide RanGTP/GDP gradient for nuclear pore complex. Importin-9 (IPO-9) is a major nucleocytoplasmic transporter for RPL18A. Among other cargos of IPO-9 are HSP27 (important anti-apoptotic player), scaffold subunit A of PP2A (major mitotic phosphatase), core histones and ribosomal proteins. Considering the essential role of nucleocytoplasmic transport in general and probable consequences of impeded transport of HSP27, PP2A and/or core histones, that actually match those observed with rigosertib, we hypothesize that binding with RPL18A in the presence of hyperphosphorylated RanGAP1•SUMO1 interferes with normal function of IPO-9, resulting in a variety of malfunctions and apoptosis observed in rigosertib treated cancer cells. Citation Format: Irina Oussenko, Saikrishna Divakar, M. V. Ramana Reddy, James F. Holland, E. Premkumar Reddy, Takao Ohnuma. RPL18A as putative target of rigosertib. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4595. doi:10.1158/1538-7445.AM2014-4595

N-Glycosidase treatment with 18O labeling and de novo sequencing argues for flagellin FliC glycopolymorphism in Pseudomonas aeruginosa

In prokaryote organisms, N-glycosylation of proteins is often correlated to cell-cell recognition and extracellular events. Those glycoproteins are potential targets for infection control. To date, many surface-glycosylated proteins from bacterial pathogens have been described. However, N-linked Pseudomonas surface-associated glycoproteins remain underexplored. We report a combined enrichment and labeling strategy to identify major glycoproteins on the outside of microorganisms. More precisely, bacteria were exposed to a mix of biotinylated lectins able to bind with glycoproteins. The latter were then recovered by avidin beads, digested with trypsin, and submitted to mass spectrometry. The targeted mixture of glycoproteins was additionally deglycosylated in the presence of H2(18)O to incorporate (18)O during PNGase F treatment and were also analyzed using mass spectrometry. This approach allowed us to identify a few tens of potential N-glycoproteins, among which flagellin FliC was the most abundant. To detect the possible sites of FliC modifications, a de novo sequencing step was also performed to discriminate between spontaneous deamidation and N-glycan loss. This approach led to the proposal of three potential N-glycosylated sites on the primary sequence of FliC: N26, N69, and N439, with two of these three asparagines belonging to an N-X-(S/T) consensus sequence. These observations suggest that flagellin FliC is a heterogeneous protein mixture containing both O- and N-glycoforms.

CDC25A-inhibitory RE derivatives bind to pocket adjacent to the catalytic site

RE derivatives, which are cell-permeable and non-electrophilic dual-specificity protein phosphatase inhibitors developed in our laboratory, inhibit CDC25A/B non-competitively, as determined by means of kinetic experiments. To identify the binding site of RE derivatives, we designed and synthesized the new probe molecule RE142, having a Michael acceptor functionality for covalent bond formation with the enzyme, a biotin tag to enable enrichment of probe-bound peptide(s), and a chemically cleavable linker to facilitate release of probe-bound peptides from avidin beads. LC-MS analysis indicated that RE142 binds to one of the residues Cys384-Tyr386 of CDC25A, within a pocket adjacent to the catalytic site.

Monolithic columns with immobilized monomeric avidin: preparation and application for affinity chromatography

A poly(glycidyl methacrylate-co-acrylamide-co-ethylene dimethacrylate) monolith and a poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith were prepared in fused silica capillaries (100 μm ID) and modified with monomeric avidin using the glutaraldehyde technique. The biotin binding capacity of monolithic affinity columns with immobilized monomeric avidin (MACMAs) was determined by fluorescence spectroscopy using biotin (5-fluorescein) conjugate, as well as biotin- and fluorescein-labeled bovine serum albumin (BSA). The affinity columns were able to bind 16.4 and 3.7 μmol biotin/mL, respectively. Columns prepared using the poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith retained 7.1 mg BSA/mL, almost six times more than commercially available monomeric avidin beads. Protocols based on MALDI-TOF mass spectrometry monitoring were optimized for the enrichment of biotinylated proteins and peptides. A comparison of enrichment efficiencies between MACMAs and commercially available monomeric avidin beads yielded superior results for our novel monolithic affinity columns. However, the affinity medium presented in this work suffers from a significant degree of nonspecific binding, which might hamper the analysis of more complex mixtures. Further modifications of the monolith's surface are envisaged for the future development of monoliths with improved enrichment characteristics.

The Histone Modifications Governing TFF1 Transcription Mediated by Estrogen Receptor

Transcription regulation by histone modifications is a major contributing factor to the structural and functional diversity in biology. These modifications are encrypted as histone codes or histone languages and function to establish and maintain heritable epigenetic codes that define the identity and the fate of the cell. Despite recent advances revealing numerous histone modifications associated with transcription regulation, how such modifications dictate the process of transcription is not fully understood. Here we describe spatial and temporal analyses of the histone modifications that are introduced during estrogen receptor α (ERα)-activated transcription. We demonstrated that aborting RNA polymerase II caused a disruption of the histone modifications that are associated with transcription elongation but had a minimal effect on modifications deposited during transcription initiation. We also found that the histone H3S10 phosphorylation mark is catalyzed by mitogen- and stress-activated protein kinase 1 (MSK1) and is recognized by a 14-3-3ζ/14-3-3ε heterodimer through its interaction with H3K4 trimethyltransferase SMYD3 and the p52 subunit of TFIIH. We showed that H3S10 phosphorylation is a prerequisite for H3K4 trimethylation. In addition, we demonstrated that SET8/PR-Set7/KMT5A is required for ERα-regulated transcription and its catalyzed H4K20 monomethylation is implicated in both transcription initiation and elongation. Our experiments provide a relatively comprehensive analysis of histone modifications associated with ERα-regulated transcription and define the biological meaning of several key components of the histone code that governs ERα-regulated transcription.

Nedd4-1 and β-Arrestin-1 Are Key Regulators of Na+/H+ Exchanger 1 Ubiquitylation, Endocytosis, and Function

The ubiquitously expressed mammalian Na(+)/H(+) exchanger 1 (NHE1) controls cell volume and pH but is also critically involved in complex biological processes like cell adhesion, cell migration, cell proliferation, and mechanosensation. Pathways controlling NHE1 turnover at the plasma membrane, however, are currently unclear. Here, we demonstrate that NHE1 undergoes ubiquitylation at the plasma membrane by a process that is unprecedented for a mammalian ion transport protein. This process requires the adapter protein β-arrestin-1 that interacts with both the E3 ubiquitin ligase Nedd4-1 and the NHE1 C terminus. Truncation of NHE1 C terminus to amino acid 550 abolishes binding to β-arrestin-1 and NHE1 ubiquitylation. Overexpression of β-arrestin-1 or of wild type but not ligase-dead Nedd4-1 increases NHE1 ubiquitylation. siRNA-mediated knock-down of Nedd4-1 or β-arrestin-1 reduces NHE1 ubiquitylation and endocytosis leading to increased NHE1 surface levels. Fibroblasts derived from β-arrestin-1 and Nedd4-1 knock-out mice show loss of NHE1 ubiquitylation, increased plasmalemmal NHE1 levels and greatly enhanced NHE1 transport compared with wild-type fibroblasts. These findings reveal Nedd4-1 and β-arrestin-1 as key regulators of NHE1 ubiquitylation, endocytosis, and function. Our data suggest a broader role for β-arrestins in the regulation of membrane ion transport proteins than currently known.

Proteomics Identification of Drosophila Small Interfering RNA-associated Factors

The Drosophila melanogaster RNA-induced silencing complex (RISC) forms a large ribonucleoprotein particle on small interfering RNAs (siRNAs) and catalyzes target mRNA cleavage during RNA interference (RNAi). Dicer-2, R2D2, Loquacious, and Argonaute-2 are examples of RISC-associated factors that are involved in RNAi. Holo-RISC is an approximately 80 S small interfering ribonucleoprotein, which suggests that there are many additional proteins that participate in the RNAi pathway. In this study, we used siRNA affinity capture combined with mass spectrometry to identify novel components of the Drosophila RNAi machinery. Our study identified both established RISC components and novel siRNA-associated factors, many of which contain domains that are consistent with potential roles in RNAi. Functional analysis of these novel siRNA-associated proteins suggests that these factors may play an important role in RNAi.

Bisaryl hydrazones as exchangeable biocompatible linkers.

The selective enrichment of tagged molecules from complex biological mixtures is of primary importance for methods in chemical biology and proteomics. Affinity purification on (strept)avidin beads using biotin as an affinity tag is one of the most widely applied methods to achieve this, fully utilizing the high affinity of biotin for (strept)avidin (Ka ≈ 1.7 × 1015 M−1)[1]. However, the method is limited by the harsh, denaturing conditions required for elution, such as an SDS boil or treatment with 8 M Guanidine (pH 1.5). Protein structure and function are lost and target-proteins may be contaminated with proteins nonspecifically bound to the beads. Two strategies have been explored to elute under mild conditions: i) weakening of the biotin-(strept)avidin interaction by modulating the Ka[2] and ii) introduction of a proteolytically[3] or chemically[4] cleavable linker. While the first strategy does improve the release of biotinylated proteins from (strept)avidin beads, it adversely affects the stringency of the immobilization. The second strategy enables site-specific cleavage, but premature cleavage has been reported and cleavage conditions have no demonstrated compatibility with active biomolecules. In addition, the general applicability of the cleavable linkers is often limited by the need for multistep organic synthesis before implementation.