Cell Free Application: Characterization of Long Non-coding RNA Inhibition of Transcription

Long noncoding RNAs have been shown to regulate chromatin states, transcriptional activity and post transcriptional activity (1). Only a few studies have observed long non-coding RNAs modulating the translational process (2). The noncoding RNA BC200 has been shown to inhibit translation by interacting with the translation initiation factors, eIF4A and eIF4B.

To characterize how BC200 translational inhibition could be controlled,  a variety of RNAs were transcribed/translated in vitro using the TNT system (Cat. #L4610) from Promega. To each transcription/translation reaction, BC900 RNA, hnRNPE1 and hnRNE2 proteins were added. Inhibition of BC200 activity was noted when proteins were successful expressed (3).

Literature Cited

  1.  Sosinska, P et.al. (2015) Intraperitoneal invasiveness of ovarian cancer from the cellular and molecular perspective. Ginekol. Pol. 86, 782–86.
  2. Geisler, S. and Coller, J. (2013) RNA in unexpected places: long non-coding RNA functions in diverse cellular contexts. Nat.Rev. Mol. Cell. Bio. 14,699–12.
  3. Jang, S. et. al. (2017) Regulation of BC200 RNA-mediated translation inhibition by hnRNP E1 and E2. FEBS Letters. 591, 393–5.

Automated Approach for Multiomic Analysis

With the use of a suite of “-omics” technologies you can examine the way in which complex cellular processes work together across all molecular domains (i.e., proteomics, metabolomics, transcriptomics) in a single biological system. Several studies have been published across a wide range of fields illustrating the power of such a unified approach (1,2). Most studies however did not focus on the development of a high-throughput, unified sample preparation approach to complement high-throughput “omic” analytics.

A recent publication by Gutierrez and colleagues presents a simple high-throughput process (SPOT) that has been optimized to provide high-quality specimens for metabolomics, proteomics, and transcriptomics from a common cell culture sample (3). They demonstrate that this approach can process  16−24 samples from a cell pellet to a desalted sample ready for mass spectrometry analysis within 9 hours. They also demonstrated that the combined process did not sacrifice the quality of data when compared to individual sample preparation methods.

Literature Cited

1. Roume, H. (2013) Sequential Isolation of Metabolites, RNA, DNA, and Proteins from the Same Unique Sample. Methods Enzymol. 531, 219−236.
2. Lo, A. W. et al. (2017) ‘Omic’ Approaches to Study Uropathogenic Escherichia Coli Virulence. Trends Microbiol. 25, 729−740.
3. Gutierrez, D. et al. (2018)  An Integrated, High-Throughput Strategy for Multiomic Systems Level Analysis J. Proteome Res.

Tetanus Neurotoxin: Potential Mechanism for Drug Delivery

Tetanus neurotoxin (TeNT), produced by Clostridium tetani, is one of the most potent neurotoxins in humans. TeNT causes tetanus, which is characterized by painful muscular contractions and spasms as well as seizure. TeNT is composed of a light chain and a heavy chain (TTH). The toxic properties of TeNT reside in the toxin light chain (L), but like complete TeNT, the TeNT heavy chain (TTH) and the C-terminal domain (TTC) alone can bind and enter into neurons.

Based on these properties, a recent publication (1) considered that TTC could be a promising vehicle to deliver drug cargos to neurons. To explore this possibility, they engineered fusion proteins containing various TeNT fragments. They chose B-cell leukemia/lymphoma 2 protein (Bcl-2) as a partner protein, because Bcl-2 is one of the most potent anti-apoptotic proteins and has an appropriate size (26kDa) to act as a fusion partner.

They tested these fusion proteins in both cell-based and cell-free protein expression systems to determine whether the purified fusion products retained both anti-apoptotic and neuronal migration properties. One construct (Bcl2-hTTC) exhibited neuronal binding and prevented cell death of neuronal PC12 cells induced by serum and NGF deprivation, as evidenced by the inhibition of cytochrome C release from the mitochondria. For in vivo assays, Bcl2-hTTC was injected into the tongues of mice and was seen to selectively migrate to hypoglossal nuclei mouse brain stems.

  1. Watanbe, Y. et. al. (2018) Tetanus toxin fragments and Bcl-2 fusion proteins : cytoprotection and retrograde axonal migration. BMC Biotechnology 18, 39.

New Recombinant Asp-N Mass Spec Protease: Improved Format and Reduced Price

Asp-N is a endoproteinase hydrolyzes peptide bonds on the N-terminal side of aspartic residues. The native form is isolated from Pseudomonas fragi. The majority of vendors currently provide a commercial product that consists of 2µg of lyophilized material in a flat bottom vial, and sold for $175–200 US. Formatting such a small amount of material in flat bottom vial can lead to inconsistent resuspension of the protease. Inconsistent working concentrations will lead to non-reproducible data. The current high price also prohibits large-scale use.

The new recombinant Asp-N protease is cloned from Stenotrophomonas maltophilia and expressed in E. coli. Recombinant Asp-N has similar amino acid cleavage specificity as compared to native Asp-N. Digestion of a yeast extract with native and recombinant Asp-N produces very similar results. Providing 10µg lyophilized material in V-shaped vial with a visible cake enables more consistent re-suspension resulting in reproducible data. Due to improved yields the list price is now approximately 40% less when compared to native enzyme.
Learn more about this new recombinant Asp-N protease.

HaloTag Application: Fluorescence Under Stress

diagram illustrating the mechanism of the stress reporterCellular stress is associated with global misfolding and aggregation of the endogenous proteome. Monitoring stress-induced abnormalities remains one of the major technical challenges facing established sensors. Misfolded monomers induced by mild stresses, however, remain largely invisible with current sensors.

In a recent publication (1) Fares and colleagues describe a new sensor based upon a fluorescent molecular rotor that is conjugated to a Halo mutant (AgHalo). In non-stressed cells, the AgHalo sensor remains largely folded, and is fluorescent when misfolded. The fluorescent molecular rotor, when conjugated to purified AgHalo to form the proteome stress sensor, is able to report on urea-induced partially unfolded (misfolded) conformations with a higher fluorescent increase than the previously reported fluorophore-based sensors. Heat-induced misfolding is also effectively monitored by the fluorescence change of the sensor that is based on fluorescent molecular rotor, but not the solvatochromic fluorophore. The unique feature of the fluorescent molecular rotor makes the new generation of the AgHalo proteome sensor more sensitive to misfolded conformations that are primarily induced by mild proteome stress. Further, the new sensor exhibits a higher fluorescence signal when detecting soluble and insoluble protein aggregates that are induced by more severe proteome stress. These data collectively suggest that thermo-labile Halo conjugated with a fluorescent molecular rotor serves as a suitable sensor to detect a wide range of proteome stress conditions.

Literature Cited

Fares, M. et al. (2018) A Molecular Rotor-Based Halo-Tag Ligand Enables a Fluorogenic Proteome Stress Sensor to Detect Protein Misfolding in Mildly Stressed Proteome. Bioconjugate Chem 29, 215–24.

Beer Is Complicated: Proteome Analysis via Mass Spectrometry

still life with a keg of beer and draft beer by the glass.The art of brewing alcoholic beverages has existed for thousands of years. The process of beer brewing begins with barley grains, which are malted to allow partial germination, triggering expression of key enzymes. The germinated grains are then dried and milled. Next, starch, proteins, and other molecules are solubilized during mashing. During mashing, solubilized enzymes degrade starch to fermentable sugars, and digest proteins to produce peptides and free amino acids. Fermentable sugars and free amino acids are required for efficient yeast growth during fermentation.

After the mash, the wort is removed, and hops are added for bitterness and aroma, and the wort is boiled. After boiling, the wort is inoculated with yeast, and fermentation proceeds to produce bright beer. Typically this bright beer is then filtered, carbonated, packaged, and sold. Many proteins originating from the barley grain and the yeast are present in beer, and these have been reported to affect the quality of the final product. However, some of the biochemical details of this process remain unclear. To better understand what happens during the various steps of the brewing process,  Schultz et al. used mass spectrometry proteomics to perform a global untargeted analysis of the proteins present across time during beer production and described this work in a recent paper (1). Samples analyzed included sweet wort produced by a high temperature infusion mash, hopped wort, and bright beer. Continue reading

Characterizing Multi-Subunit Protein Complexes Using Cell-Free Expression

artist's concept of a cell membraneMulti-subunit protein complexes control membrane fusion events in eukaryotic cells (1). CORVET and HOPS are two such multi-subunit complexes, both containing the Sec1/Munc18 protein subunit VPS33A (2). Metazoans additionally possess VPS33B, which has considerable sequence similarity to VPS33A but does not integrate into CORVET or HOPS complexes and instead stably interacts with VIPAR. Recent research suggests that VPS33B and VIPAR comprise two subunits of a novel multi-subunit complex analogous in configuration to CORVET and HOPS (3).

In a recent publication (4), Hunter and colleagues, further characterized the VPS33B and VIPAR complex. Using co-immunoprecipitation and proximity-based ligation assay, they identified two novel VPS33B-interacting proteins, VPS53 and CCDC22.

In vitro binding experiments, VPS33B and GST-VIPAR were co-expressed in Escherichia coli and purified by GSH affinity. The VPS33B/GSTVIPAR complex was used as bait in pulldown experiments, with myc-CCDC22 and myc-VPS53 expressed by cell-free in vitro transcription/translation in wheat germ lysate. Myc-CCDC22 was very efficiently pulled down by VPS33B/GST-VIPAR, whereas myc-VPS53 was not .The interaction between VPS53 and the VPS33B-VIPAR complex was either indirect, requires other proteins contribute to the interaction, or requires a post-translational modification not conferred in the plant cell-free expression system (wheat germ). Pull-down experiments with individual subunits or expressing as complexes, was inefficient and did not result in binding to VPS33B/GST-VIPAR.

To further understand how VPS33B-VIPAR may interact with CCDC22, Hunter and colleagues attempted to refine the region of CCDC22 that interacts with VPS33B/GST-VIPAR by generating a series of truncated forms of CCDC22. However, none of five CCDC22 truncations were able to bind to VPS33B/GST-VIPAR. The hypothesis was that truncated forms of CCDC22 are unstable and unable to fold correctly in this assay system.

Additional experiments noted that the protein complex in HEK293T cells which contained VPS33B and VIPAR was considerably smaller than CORVET/HOPS, suggesting that, unlike VPS33A, VPS33B does not assemble into a large stable multi-subunit protein complex.

 

  1. D’Agostino, M. et. al. (2017) A tethering complex drives the terminal stage of SNARE-dependent membrane fusion. Nature 551, 634–638.
  2. Balderhaar, H. J. K. and Ungermann, C. (2013) CORVET and HOPS tethering complexes – coordinators of endosome and lysosome fusion. J. Cell Sci. 126, 1307–16.
  3. Spang, A. (2016) Membrane Tethering Complexes in the Endosomal System. Front. Cell Dev. Biol. 4, 35.
  4. Hunter, M.  et. al.  (2017) Proteomic and biochemical comparison of the cellular interaction partners of human VPS33A and VPS33B. [Internet bioRxiv http://dx.doi.org/10.1101/236695  Accessed 3/12/2018]

Mass Spec Analysis of PTMs Using Minimal Sample Material

DNA is organized by protein:DNA complexes called nucleosomes in eukaryotes. Nucleosomes are composed of 147 base pairs of DNA wrapped around a histone octamer containing two copies of each core histone protein. Histone proteins play significant roles in many nuclear processes including transcription, DNA damage repair and heterochromatin formation. Histone proteins are extensively and dynamically post-translationally modified, and these post-translational modifications (PTMs) are thought to comprise a specific combinatorial PTM profile of a histone that dictates its specific function.  Abnormal regulations of PTM may lead to developmental disorders and disease development such as cancer.

Antibodies have been widely used to characterize histones and histone PTMs. However, antibody-based techniques have several limitations. Mass spectrometry (MS) has therefore emerged as the most suitable analytical tool to quantify proteomes and protein PTMs.  The most commonly used strategy is still bottom-up MS, and the most widely adopted protocol includes derivatization of lysine residues in histones to allow trypsin to generate Arg-C like peptides (4–20 aa). However, samples such as primary tissues, complex model systems, and biofluids are hard to retrieve in large quantities. Because of this, it is critical to know whether the amount of sample available would lead to an exhaustive analysis if subjected to MS.

In a recent publication, Guo, et al. examined (1) the reproducibility in quantification of histone PTMs using a wide range of starting material: from 50,000 to 5,000,000 cells. They used four different cell lines: HeLa, 293T, human embryonic stem cells (hESCs), and myoblasts. Their results demonstrated that an accurate quantification of abundant histone PTMs can be efficiently obtained by using low-resolution MS and as low as 50,000 cells as starting material Low abundance histone marks showed more variability in quantification when comparing different amounts of starting material, so a larger amount of starting material (at least 500,000 cells) is recommended.

Reference

Guo, Q. et al. (2017) Assessment of Quantification Precision of Histone Post-Translational Modifications by Using an Ion Trap and down To 50,000 Cells as Starting Material. J. Proteome Res. 17, 234–42.

Optimized Detection of EPO-Fc in Human Biological Fluids

Recombinant erythropoietin (rhEPO) is often used as “doping agent” by athletes in endurance sports to increase blood oxygen capacity. Some strategies improve the pharmacological properties of erythropoietin (EPO) through the genetic and chemical modification of the native EPO protein. The EPO-Fcs are fusion proteins composed of monomeric or dimeric recombinant EPO and the dimeric Fc region of human IgG molecules. The Fc region includes the hinge region and the CH2 and CH3 domains. Recombinant human EPOs (rhEPO) fused to the IgG Fc domain demonstrate a prolonged half-life and enhanced erythropoietic activity in vivo compared with native or rhEPO.

Drug-testing agencies will need to obtain primary structure information and develop a reliable analytical method for the determination of EPO-Fc abuse in sport. The possibility of EPO-Fc detection using nanohigh-performance liquid chromatography−tandem mass spectrometry (HPLC−MS/MS) was already demonstrated (1). However, the prototyping peptides derived from EPO and IgG are not selective enough because both free proteins are naturally presented in human serum. In a recent publication, researchers describe the effort to identify peptides covering unknown fusion breakpoints (later referred to as “spacer” peptides; 2). The identification of “spacer” peptides will allow the confirmation of the presence of exogenous EPO-Fc in human biological fluids.

A bottom-up approach and the intact molecular weight measurement of deglycosylated protein and its IdeS proteolytic fractions was used to determine the amino acid sequence of EPO-Fc. Using multiple proteases, peptides covering unknown fusion breakpoints (spacer peptides) were identified.

Results indicated that “spacer peptides” could be used in the determination of EPO-Fc fusion proteins in biological samples using common LC−tandem MS methods.

References

  1. Reichel, C. et al. (2012) Detection of EPO-Fc fusion protein in human blood: screening and confirmation protocols for sports drug testing.
    Drug Test. Anal. 4, 818−29.
  2. Mesonzhnik, N. et al. (2017) Characterization and Detection of Erythropoietin Fc Fusion Proteins Using Liquid Chromatography−Mass Spectrometry.
    J. of Proteome Res. 17, 689-97.

Luciferase Immunoprecipitation System Assay (LIPS): Expression of Luciferase Antigen using TNT Transcription/Translation Kit

NanoLuc dual reporters

Illustration showing NanoLuc and firefly luciferase reporters.

The luciferase immunoprecipitation system (LIPS) assay is a liquid phase immunoassay allowing high-throughput serological screening of antigen-specific antibodies. The immunoassay involves quantitating serum antibodies by measuring luminescence emitted by the reporter enzyme Renilla luciferase (Rluc) fused to an antigen of interest. The Rluc-antigen fusion protein is recognized by antigen-specific antibodies, and antigen-antibody complexes are captured by protein A/G beads that recognize the Fc region of the IgG antibody (1).

In a recent publication (2), this assay was used to assess the presence of autoantibodies against ATP4A and ATP4B subunits of parietal cells H+, K+-ATPase in patients with atrophic body gastritis and in controls. Continue reading