We would like to thank Dr. David Engelke for his guidance and helpful comments on this manuscript. PJS - Ball State University laboratory startup funds; DAB - grant 1R15AI130950-01.

NOTE: The protocol was adapted from Ref. 24.
1. Obtain RNA and Enzyme of Interest
<ol>
	<li>Use in vitro transcribed RNAs internally labeled with 32P, and recombinant His6-Mod5 expressed in and purified from E. coli, as previously described24.
	<ol>
		<li>Introduce in vitro transcribed RNAs (section 3) using T7 RNA polymerase in the presence of unlabeled ATP, UTP, CTP, GTP and 10 &#181;Ci of gel-purified, ethanol-precipitated &#945;-ATP, resuspended in 1x TE (10 mM Tris-HCl pH 7.5, 0.1 mM EDTA), and stored at -80 &#176;C24.</li>
	</ol>
	</li>
	<li>Alternatively, obtain commercially available fluorescently labeled RNAs of interest. Produce the enzyme(s) of interest in any preferred expression system. 
	Caution: Internal fluorescent tags in the RNA can alter RNA structure and recognition by enzymes.</li>
</ol>
2. Prepare a 20% Polyacrylamide Denaturing Gel
Note: In order to obtain sufficient resolution of RNA fragments, a 40 cm length vertical slab gel is recommended. The width of the gel used is determined by the number of samples to be analyzed.
<ol>
	<li>Thoroughly clean glass plates. First, wash with soap and water, rinse well with deionized water, and finally clean with isopropanol and lint-free wipes.</li>
	<li>Assemble the plates and spacers.</li>
	<li>Mix the following reagents to make 100 mL of 20% acrylamide, 7.5 M urea, 1x TBE gel: 80 mL of urea gel concentrate (237.5 g/L of acrylamide, 12.5 g/L of methylene bisacrylamide, 7.5 M urea in deionized water), 10 mL of urea gel diluent (7.5M urea in deionized water), and 10 mL of urea gel buffer (0.89 M Tris-Borate-20 mM EDTA buffer pH 8.3 and 7.5 M urea). 
	NOTE: The volume of gel solution must be adjusted according to the dimensions of gel.</li>
	<li>Add 40 &#181;L of N,N,N&#8242;,N&#8242;-Tetramethylethylenediamine (TEMED) and 800 &#181;L of freshly prepared 10% ammonium persulfate (APS).</li>
	<li>Draw gel solution up into a large syringe and dispense between glass plates. Tap on glass with fingers while pouring to prevent bubble formation.</li>
	<li>Allow gel to solidify for 30 min.</li>
	<li>Clamp solidified gel onto vertical gel apparatus using binder clips.</li>
	<li>Fill upper and lower buffer chambers with 1x TBE.</li>
	<li>Two hours prior to loading the gel, pre-run the gel at 20 mA, for 2 h to allow the buffer boundary to outrun the smallest oligonucleotides - otherwise the smallest nucleotides and oligonucleotides tend to collapse at the buffer front.</li>
</ol>
3. RNA Isopentenylation Assay
<ol>
	<li>Prepare reactions in a final volume of 17 &#181;L, containing 58 mM Tris-HCl (pH 7.2), 1.2 mM ATP, 5.8 mM MgCl2, 0.2 mM DMAPP, 10 U of RNase inhibitor (e.g., SuperRNaseIn), 40,000 CPM of internally 32P-labeled RNA, 5.3 &#181;M Mod5, and 1.2 mM 2-mercaptoethanol.</li>
	<li>Incubate reactions at 37 &#176;C for 1 h.</li>
	<li>Ethanol-precipitate RNAs using 2.5 volumes (42.5 &#181;L) of 100% ethanol and 1/10 volumes (1.7 &#181;L) of 3.5 M sodium acetate pH 5.5, and place at -20 &#176;C for 1 h or overnight.</li>
	<li>Centrifuge RNA samples for 20 min at 15,400 x g and 4 &#176;C.</li>
	<li>Carefully remove the supernatant and wash the RNA pellet with 500 &#181;L of 70% ethanol.</li>
	<li>Centrifuge samples at for 5 min 15,400 x g and 4&#176;C.</li>
	<li>Carefully remove the supernatant and air-dry RNA pellets for 15 min, or until all ethanol has evaporated.</li>
	<li>Resuspend RNA pellets in 10 &#181;L of 8 M urea.</li>
	<li>Add 150 U of RNase T1 and incubate at 37 &#176;C overnight.</li>
	<li>Add 2 &#181;L of 6x loading buffer (60% glycerol, 0.1% xylene cyanol).</li>
	<li>Load 10 &#181;L of each RNA sample on a pre-run, 20% polyacrylamide, 7.5 M urea gel (see section 2 of Protocol). 
	NOTE: Radiolabeled RNA size ladders may be included as an additional mobility marker.</li>
	<li>Run gel for 2 h at 25 mA.</li>
	<li>Stop gel and remove from apparatus.</li>
	<li>Break seal between the two glass plates and remove one of the glass plates, with the gel remaining on the &#34;bottom&#34; plate. 
	NOTE: Take care not to tear the gel during this step.</li>
	<li>Place a layer of plastic wrap over the gel and expose on a phosphor screen for 3 h. Alternatively, place gel on chromatography paper and dry with a gel dryer prior to phosphor screen exposure.</li>
	<li>Image phosphor screen on a phosphor imager.</li>
</ol>

<ol>
	<li>Boccaletto, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MODOMICS:+a+database+of+RNA+modification+pathways.+2017+update.">MODOMICS: a database of RNA modification pathways. 2017 update.</a> Nucleic Acids Research. 46 (D1), 303-307 (2018).</li><li>Lewis, C. 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C., Esberg, B., Olafsson, O., Bjork, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+function+of+isopentenyl+adenosine+derivatives+in+tRNA.">Synthesis and function of isopentenyl adenosine derivatives in tRNA.</a> Biochimie. 76 (12), 1152-1160 (1994).</li><li>Urbonavicius, J., Qian, Q., Durand, J. M., Hagervall, T. G., Bjork, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improvement+of+reading+frame+maintenance+is+a+common+function+for+several+tRNA+modifications.">Improvement of reading frame maintenance is a common function for several tRNA modifications.</a> EMBO Journal. 20 (17), 4863-4873 (2001).</li><li>Aubee, J. I., Olu, M., Thompson, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+i6A37+tRNA+modification+is+essential+for+proper+decoding+of+UUX-Leucine+codons+during+rpoS+and+iraP+translation.">The i6A37 tRNA modification is essential for proper decoding of UUX-Leucine codons during rpoS and iraP translation.</a> RNA. 22 (5), 729-742 (2016).</li><li>Caillet, J., Droogmans, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+cloning+of+the+Escherichia+coli+miaA+gene+involved+in+the+formation+of+delta+2-isopentenyl+adenosine+in+tRNA.">Molecular cloning of the Escherichia coli miaA gene involved in the formation of delta 2-isopentenyl adenosine in tRNA.</a> Journal of Bacteriology. 170 (9), 4147-4152 (1988).</li><li>Soderberg, T., Poulter, C. 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J., Rusconi, C. P., White, I. E., Faust, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+sequencing+of+two+isopentenyladenosine-modified+transfer+RNAs+from+Chinese+hamster+ovary+cells.">Identification and sequencing of two isopentenyladenosine-modified transfer RNAs from Chinese hamster ovary cells.</a> Nucleic Acids Research. 26 (23), 5533-5535 (1998).</li><li>Golovko, A., Hjalm, G., Sitbon, F., Nicander, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+of+a+human+tRNA+isopentenyl+transferase.">Cloning of a human tRNA isopentenyl transferase.</a> Gene. 258 (1-2), 85-93 (2000).</li><li>Kernohan, K. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matchmaking+facilitates+the+diagnosis+of+an+autosomal-recessive+mitochondrial+disease+caused+by+biallelic+mutation+of+the+tRNA+isopentenyltransferase+(TRIT1)+gene.">Matchmaking facilitates the diagnosis of an autosomal-recessive mitochondrial disease caused by biallelic mutation of the tRNA isopentenyltransferase (TRIT1) gene.</a> Human Mutations. 38 (5), 511-516 (2017).</li><li>Yarham, J. 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None to disclose.

RNA modifications continue to be shown to play ever more important and diverse roles in cellular and organismal function. As such, the development of assays to interrogate RNA modifying enzymes is central to better understanding the fundamental aspects of biology. This protocol describes a high-resolution in vitro assay to characterize the tRNA modification activity of Mod5.
This protocol has the distinct advantage of providing a direct, and easily interpretable readout of isopentenyl...

At least 163 distinct posttranscriptional RNA modifications have been identified, with these modifications conferring diverse and context-dependent functions to RNAs, directly influencing RNA structure, and affecting interactions of RNA with other molecules1,2. As the appreciation for the number and variety of RNA modifications increases, it is critical to develop assays that can reliably interrogate both the RNA modifications and the enzymes that catalyze them.
One of the first RNA modifications to be identified occurs at base 37 in tRNAs, adjacent to the anti-codon on the 3&#39; side3,4. An isopentenyl group is transferred from dimethylallylpyrophosphate (DMAPP) to the N6 position of adenosine 37 (i6A37) 3,5 on a subset of both cytoplasmic and mitochondrial tRNAs. i6A37 improves translation fidelity and efficiency by increasing the tRNA&#39;s affinity for the ribosome4,6 and i6A37 is important for stress response in bacteria7. The enzymes that perform this modification are termed tRNA isopentenyl transferases and are highly conserved in bacteria8,9, fungi10, worms11, plants12, and higher eukaryotes13, including humans14.
Mutations in the human tRNA isopentenyl transferase gene, TRIT1, are associated with human disease. For example, a mutation in TRIT1 is correlated with a severe mitochondrial disease, likely caused by a defect in mitochondrial protein synthesis15,16. Furthermore, TRIT1 has been described as a tumor suppressor gene17,18 and is implicated in several types of cancers including melanoma19, breast20, gastric21, and lung cancers22,23. Finally, TRIT1 and Mod5 (Saccharomyces cerevisiae) isopentenyl transferases are aggregation-prone proteins that form prion-like amyloid fibers24,25,26. These observations potentially implicate tRNA isopentenyl transferases in neurodegenerative diseases, although direct evidence for this has not yet been shown.
Given the role that isopentenyl transferases play in translation and disease, methods that directly measure i6A isopentenyl transferase activity are important for a mechanistic understanding of these enzymes under normal and disease states. An increasing number of methods are available to detect i6A RNA modifications, including in vitro isopentenylation assays, positive hybridization in the absence of i6A (PHA6) assays, thin layer chromatography (TLC), amino acid acceptance activity assays, and mass spectrometry approaches (Reviewed in Ref. 4).
An in vitro isopentenylation assay has been described that utilizes 14C-DMAPP and unlabeled tRNAs. In this assay, radioactive carbon is transferred to RNA from 14C-DMAPP by the isopentenyl transferase. While this assay is highly sensitive, it is often difficult to determine the specific residue that is modified9,20,27. PHA6 assays rely on the bulky i6A modification interfering with hybridization of a 32P-labeled probe spanning the modified residue. As such, hybridization is greater in the absence of an i6A modification18,28,29. PHA6 assays are highly sensitive, and capable of analyzing total RNA extracted from cellular lysates. Additionally, the ability to design probes specific to the RNA of interest gives this method substantial target flexibility. However, PHA6 assays are limited to the characterization of modifications that occur on residues within the targeted region of the probe and therefore are less likely to identify novel modification sites. In addition, as absence of binding is indicative of modification, other modifications or mutations that affect RNA binding will confound data analysis.
Another approach combines benzyl DEAE cellulose (BD) cellulose chromatography with amino acid acceptance activity as a readout of i6A modifications in tRNA30. This approach directly assays the function of the i6A modification, but it is an indirect approach to detect i6A modification and lacks resolution to map modifications to a specific residue in the RNA. A TLC approach has been used to detect total tRNA i6A modifications. In this approach, internally 32P-labeled tRNAs are digested to single nucleotides and two-dimensional TLC analysis is used to identify isopentenylation. This approach is highly sensitive in detecting total i6A in a given RNA sample but upon digestion, all sequence information is lost; thus, the investigator has no way of determining which residues have been modified31.
More recently, liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods have been developed that quantitatively compare total RNA modifications between species, cell types, and experimental conditions32,33,34. A limitation of this methodology is that it is less able to determine the identity and position within the RNA from which the modified nucleoside was derived34. Furthermore, the expertise and equipment necessary to execute these experiments limit the practicality of this approach.
In addition, several next-generation sequencing technologies have been developed to map RNA modifications transcriptome-wide34. Immunoprecipitation of RNAs with antibodies specific to a particular modification (RIP-Seq) enable the investigator to identify all sequences containing a specific modification35,36. Additionally, reverse transcriptase-based approaches such as Chem-Seq and non-random mismatch sequencing rely on perturbations of the reverse transcription reaction at the modified residues37,38,39. Despite the advantage of these techniques to map RNA modifications transcriptome-wide, RIP-seq and Chem-Seq technologies are limited by the lack of reliable antibodies or reactive chemicals available for each specific modification, respectively34. Furthermore, reverse transcriptase enzymes required to perform Chem-Seq and non-random mismatch sequencing techniques can be impeded by stable RNA structures. The highly modified and structurally stable nature of tRNAs make them especially difficult to interrogate using these techniques. To date, next-generation sequencing-based technologies have not yet been utilized to map i6A modifications34.
Herein, we describe a simple and direct approach to detect i6A tRNA modifications in vitro. This method utilizes incubation of RNAs with recombinant S. cerevisiae isopentenyl transferase (Mod5), followed by RNase T1 digestion, and 1-dimensional gel electrophoresis analysis to map i6A modifications. This approach is direct and requires little specialized expertise to analyze the data. Furthermore, this method is adaptable to other RNA modifying enzymes, including enzymes that covalently change the molecular weight of RNA or an RNA&#39;s mobility through a gel.

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an in vitro assay to detect trna-isopentenyl transferase activity

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved N6-isopentenyladenosine modifications occurs at the A37 position in a subset of tRNAs. This modification improves translation efficiency and fidelity by increasing the affinity of the tRNA for the ribosome. Mutation of enzymes responsible for this modification in eukaryotes are associated with several disease states, including mitochondrial dysfunction and cancer. Therefore, understanding the substrate specificity and biochemical activities of these enzymes is important for understanding of normal and pathologic eukaryotic biology. A diverse array of methods has been employed to characterize i6A modifications. Herein is described a direct approach for the detection of isopentenylation by Mod5. This method utilizes incubation of RNAs with a recombinant isopentenyl transferase, followed by RNase T1 digestion, and 1-dimensional gel electrophoresis analysis to detect i6A modifications. In addition, the potential adaptability of this protocol to characterize other RNA-modifying enzymes is discussed.

Mod5 was incubated with a tyrosine tRNA or serine tRNA in the presence or absence of DMAPP. Following the modification reaction, products were RNase T1-digested, which cleaves the 3&#39; end of all guanosines leaving a 3&#39; guanosine monophosphates (GMP)24 (Figure 1). Full digestion of the RNAs produces a predictable pattern of radiolabeled fragments (Figure 2A), which are then resolved ...

Watch this Scientific Journal Video about An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity at JoVE.com

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

Here, we describe a protocol for the biochemical characterization of the yeast RNA-modifying enzyme, Mod5, and discuss how this protocol could be applied to other RNA-modifying enzymes.

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved ...

an-in-vitro-assay-to-detect-trna-isopentenyl-transferase-activity

Research

JoVE Journal

Biochemistry

6.9K Views.  Ball State University. Here, we describe a protocol for the biochemical characterization of the yeast RNA-modifying enzyme, Mod5, and discuss how this protocol could be applied to other RNA-modifying enzymes.

Video: An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many of these conditions cause protein unfolding in the cell and have detrimental impact on the survival of the organism. A group of unrelated, stress-specific molecular chaperones have been shown to play essential roles in the survival of these stress conditions. While fully folded and chaperone-inactive before stress, these proteins rapidly unfold and become chaperone-active under specific stress conditions. Once activated, these conditionally disordered chaperones bind to a large number of different aggregation-prone proteins, prevent their aggregation and either directly or indirectly facilitate protein refolding upon return to non-stress conditions. The primary approach for gaining a more detailed understanding about the mechanism of their activation and client recognition involves the purification and subsequent characterization of these proteins using in vitro chaperone assays. Follow-up in vivo stress assays are absolutely essential to independently confirm the obtained in vitro results.
This protocol describes in vitro and in vivo methods to characterize the chaperone activity of E. coli HdeB, an acid-activated chaperone. Light scattering measurements were used as a convenient read-out for HdeB&#39;s capacity to prevent acid-induced aggregation of an established model client protein, MDH, in vitro. Analytical ultracentrifugation experiments were applied to reveal complex formation between HdeB and its client protein LDH, to shed light into the fate of client proteins upon their return to non-stress conditions. Enzymatic activity assays of the client proteins were conducted to monitor the effects of HdeB on pH-induced client inactivation and reactivation. Finally, survival studies were used to monitor the influence of HdeB&#39;s chaperone function in vivo.

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

This study describes biophysical, biochemical and molecular techniques to characterize the chaperone activity of Escherichia coli HdeB under acidic pH conditions. These methods have been successfully applied for other acid-protective chaperones such as HdeA and can be modified to work for other chaperones and stress conditions.

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many ...

A Fluorescence-based Assay of Phospholipid Scramblase Activity

Scramblases translocate phospholipids across the membrane bilayer bidirectionally in an ATP-independent manner. The first scramblase to be identified and biochemically verified was opsin, the apoprotein of the photoreceptor rhodopsin. Rhodopsin is a G protein-coupled receptor localized in rod photoreceptor disc membranes of the retina where it is responsible for the perception of light. Rhodopsin&#39;s scramblase activity does not depend on its ligand 11-cis-retinal, i.e., the apoprotein opsin is also active as a scramblase. Although constitutive and regulated phospholipid scrambling play an important role in cell physiology, only a few phospholipid scramblases have been identified so far besides opsin. Here we describe a fluorescence-based assay of opsin&#39;s scramblase activity. Opsin is reconstituted into large unilamellar liposomes composed of phosphatidylcholine, phosphatidylglycerol and a trace quantity of fluorescent NBD-labeled PC (1-palmitoyl-2-{6-[7-nitro-2-1,3-benzoxadiazole-4-yl)amino]hexanoyl}-sn-glycero-3-phosphocholine). Scramblase activity is determined by measuring the extent to which NBD-PC molecules located in the inner leaflet of the vesicle are able to access the outer leaflet where their fluorescence is chemically eliminated by a reducing agent that cannot cross the membrane. The methods we describe have general applicability and can be used to identify and characterize scramblase activities of other membrane proteins.

We describe a fluorescence-based assay to measure phospholipid scrambling in large unilamellar liposomes reconstituted with opsin.

Scramblases translocate phospholipids across the membrane bilayer bidirectionally in an ATP-independent manner. The first scramblase to be identified and ...

Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase

Activity-based protein profiling (ABPP) is a method for the identification of an enzyme of interest in a complex proteome through the use of a chemical probe that targets the enzyme&#39;s active sites. A reporter tag introduced into the probe allows for the detection of the labeled enzyme by in-gel fluorescence scanning, protein blot, fluorescence microscopy, or liquid chromatography-mass spectrometry. Here, we describe the preparation and use of the compound ARN14686, a click chemistry activity-based probe (CC-ABP) that selectively recognizes the enzyme N-acylethanolamine acid amidase (NAAA). NAAA is a cysteine hydrolase that promotes inflammation by deactivating endogenous peroxisome proliferator-activated receptor (PPAR)-alpha agonists such as palmitoylethanolamide (PEA) and oleoylethanolamide (OEA). NAAA is synthesized as an inactive full-length proenzyme, which is activated by autoproteolysis in the acidic pH of the lysosome. Localization studies have shown that NAAA is predominantly expressed in macrophages and other monocyte-derived cells, as well as in B-lymphocytes. We provide examples of how ARN14686 can be used to detect and quantify active NAAA ex vivo in rodent tissues by protein blot and fluorescence microscopy.

Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase

Here, we describe the preparation and use of an activity-based probe (ARN14686, undec-10-ynyl-N-[(3S)-2-oxoazetidin-3-yl]carbamate) that allows for the detection and quantification of the active form of the proinflammatory enzyme N-acylethanolamine acid amidase (NAAA), both in vitro and ex vivo.

Activity-based protein profiling (ABPP) is a method for the identification of an enzyme of interest in a complex proteome through the use of a chemical ...

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; however, the number of identified targets for Cdk1, particularly in human cells is still low. The identification of Cdk1-specific phosphorylation sites is important, as they provide mechanistic insights into how Cdk1 controls the cell cycle. Cell cycle regulation is critical for faithful chromosome segregation, and defects in this complicated process lead to chromosomal aberrations and cancer.
Here, we describe an in vitro kinase assay that is used to identify Cdk1-specific phosphorylation sites. In this assay, a purified protein is phosphorylated in vitro by commercially available human Cdk1/cyclin B. Successful phosphorylation is confirmed by SDS-PAGE, and phosphorylation sites are subsequently identified by mass spectrometry. We also describe purification protocols that yield highly pure and homogeneous protein preparations suitable for the kinase assay, and a binding assay for the functional verification of the identified phosphorylation sites, which probes the interaction between a classical nuclear localization signal (cNLS) and its nuclear transport receptor karyopherin &#945;. To aid with experimental design, we review approaches for the prediction of Cdk1-specific phosphorylation sites from protein sequences. Together these protocols present a very powerful approach that yields Cdk1-specific phosphorylation sites and enables mechanistic studies into how Cdk1 controls the cell cycle. Since this method relies on purified proteins, it can be applied to any model organism and yields reliable results, especially when combined with cell functional studies.

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Cyclin-dependent kinase 1 (Cdk1) is activated in the G2 phase of the cell cycle and regulates many cellular pathways. Here, we present a protocol for an in vitro kinase assay with Cdk1, which allows the identification of Cdk1-specific phosphorylation sites for establishing cellular targets of this important kinase.

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; ...

Quantification of Hypopigmentation Activity In Vitro

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is synthesized in response to multiple cellular and environmental factors. Melanin protects skin cells from ultraviolet damage, but also has biophysical and biochemical functions. Excessive production or accumulation of melanin in melanocytes can cause dermatological problems, such as freckles, dark spots, melasma, and moles. Therefore, the control of melanogenesis with hypopigmentation agents is important in individuals with clinical or cosmetic needs. Melanin is primarily synthesized in the melanosomes of melanocytes in a complex biochemical process called melanogenesis, which is influenced by extrinsic and intrinsic factors, such as hormones, inflammation, age, and ultraviolet light exposure. We describe three methods to determine the hypopigmentation activity of chemicals or natural substances in melanocytes: measurement of the 1) cellular tyrosinase activity and 2) melanin content, and 3) staining and quantifying cellular melanin with image analysis.
In melanogenesis, tyrosinase catalyzes the rate-limiting step that converts L-tyrosine into 3,4-dihydroxyphenylalanine (L-DOPA) and then into dopaquinone. Therefore, the inhibition of tyrosinase is a primary hypopigmentation mechanism. In cultured melanocytes, tyrosinase activity can be quantified by adding L-DOPA as a substrate and measuring dopaquinone production by spectrophotometry. Melanogenesis can also be measured by quantifying the melanin content. The melanin-containing cellular fraction is extracted with NaOH and melanin is quantified spectrophotometrically. Finally, the melanin content can be quantified by image analysis following Fontana-Masson staining of melanin. Although the results of these in vitro assays may not always be reproduced in human skin, these methods are widely used in melanogenesis research, especially as the initial step to identify potential hypopigmentation activity. These methods can also be used to assess melanocyte activity, growth, and differentiation. Consistent results with the three different methods ensure the validity of the effects.

We describe three experimental methods for evaluating the hypopigmentation activity of chemicals in vitro: quantification of 1) cellular tyrosinase activity and 2) melanin content, and 3) measurement of melanin by cellular melanin staining and image analysis.

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is ...

A Colorimetric Assay of Citrate Synthase Activity in Drosophila Melanogaster

Mitochondria play the most prominent roles in cellular metabolism by producing ATP through oxidative phosphorylation and regulating a variety of physiological processes. Mitochondrial dysfunction is a primary cause of a number of metabolic and neurodegenerative diseases. Intact mitochondria are critical for their proper functioning. The enzyme citrate synthase is localized in the mitochondrial matrix and thus can be used as a quantitative enzyme marker of intact mitochondrial mass. Given that many molecules and pathways that have important functions in mitochondria are highly conserved between humans and Drosophila, and that an array of powerful genetic tools are available in Drosophila, Drosophila serves as a good model system for studying mitochondrial function. Here, we present a protocol for fast and simple measurement of citrate synthase activity in tissue homogenate from adult flies without isolating mitochondria. This protocol is also suitable for measuring citrate synthase activity in larvae, cultured cells, and mammalian tissues.

A Colorimetric Assay of Citrate Synthase Activity in Drosophila Melanogaster

We present a protocol for a colorimetric assay of citrate synthase activity for quantification of intact mitochondrial mass in Drosophila tissue homogenates.

Mitochondria play the most prominent roles in cellular metabolism by producing ATP through oxidative phosphorylation and regulating a variety of ...

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

Cap-dependent protein synthesis is the predominant translation pathway in eukaryotic cells. While various biochemical and genetic approaches have allowed extensive studies of cap-dependent translation and its regulation, high resolution kinetic characterization of this translation pathway is still lacking. Recently, we developed an in vitro assay to measure cap-dependent translation kinetics with single-molecule resolution. The assay is based on fluorescently labeled antibody binding to nascent epitope-tagged polypeptide. By imaging the binding and dissociation of antibodies to and from nascent peptide&#8211;ribosome&#8211;mRNA complexes, the translation progression on individual mRNAs can be tracked. Here, we present a protocol for establishing this assay, including mRNA and PEGylated slide preparations, real-time imaging of translation, and analysis of single molecule trajectories. This assay enables tracking of individual cap-dependent translation events and resolves key translation kinetics, such as initiation and elongation rates. The assay can be widely applied to distinct translation systems and should broadly benefit in vitro studies of cap-dependent translation kinetics and translational control mechanisms.

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

Tracking individual translation events allows for high-resolution kinetic studies of cap-dependent translation mechanisms. Here we demonstrate an in vitro single-molecule assay based on imaging interactions between fluorescently labeled antibodies and epitope-tagged nascent peptides. This method enables single-molecule characterization of initiation and peptide elongation kinetics during active in vitro cap-dependent translation.

Cap-dependent protein synthesis is the predominant translation pathway in eukaryotic cells. While various biochemical and genetic approaches have allowed ...

A High-Throughput Enzyme-Coupled Activity Assay to Probe Small Molecule Interaction with the dNTPase SAMHD1

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a pivotal regulator of intracellular deoxynucleoside triphosphate (dNTP) pools, as this enzyme can hydrolyze dNTPs into their corresponding nucleosides and inorganic triphosphates. Due to its critical role in nucleotide metabolism, its association to several pathologies, and its role in therapy resistance, intense research is currently being carried out for a better understanding of both the regulation and cellular function of this enzyme. For this reason, development of simple and inexpensive high-throughput amenable methods to probe small molecule interaction with SAMHD1, such as allosteric regulators, substrates, or inhibitors, is vital. To this purpose, the enzyme-coupled malachite green assay is a simple and robust colorimetric assay that can be deployed in a 384-microwell plate format allowing the indirect measurement of SAMHD1 activity. As SAMHD1 releases the triphosphate group from nucleotide substrates, we can couple a pyrophosphatase activity to this reaction, thereby producing inorganic phosphate, which can be quantified by the malachite green reagent through the formation of a phosphomolybdate malachite green complex. Here, we show the application of this methodology to characterize known inhibitors of SAMHD1 and to decipher the mechanisms involved in SAMHD1 catalysis of non-canonical substrates and regulation by allosteric activators, exemplified by nucleoside-based anticancer drugs. Thus, the enzyme-coupled malachite green assay is a powerful tool to study SAMHD1, and furthermore, could also be utilized in the study of several enzymes which release phosphate species.

SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase with critical roles in human health and disease. Here we present a versatile enzyme-coupled SAMHD1 activity assay, deployed in a 384-well microplate format, that allows for the evaluation of small molecules and nucleotide analogues as SAMHD1 substrates, activators, and inhibitors.

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a pivotal regulator of intracellular deoxynucleoside triphosphate (dNTP) pools, as this ...

Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) establish and maintain the DNA methylation pattern. Changes to the normal DNA methylation pattern are linked to cancer development and progression, making DNMTs potential cancer drug targets. Thus, identifying and characterizing novel small molecule inhibitors of these enzymes is of great importance. This paper presents a protocol that can be used to screen for DNA methyltransferase inhibitors. The continuous coupled kinetics assay allows for initial velocities of DNA methylation to be determined in the presence and absence of potential small molecule inhibitors. The assay uses the methyl-sensitive endonuclease Gla I to couple methylation of a hemimethylated DNA substrate to fluorescence generation.
This continuous assay allows for enzyme activity to be monitored in real time. Conducting the assay in small volumes in microtiter plates reduces the cost of reagents. Using this assay, a small example screen was conducted for inhibitors of DNMT1, the most abundant DNMT isozyme in humans. The highly substituted anthraquinone natural product, laccaic acid A, is a potent, DNA-competitive inhibitor of DNMT1. Here, we examine three potential small molecule inhibitors &#8212; anthraquinones or anthraquinone-like molecules with one to three substituents &#8212; at two concentrations to describe the assay protocol. Initial velocities are used to calculate the percent activity observed in the presence of each molecule. One of three compounds examined exhibits concentration-dependent inhibition of DNMT1 activity, indicating that it is a potential inhibitor of DNMT1.

DNA methyltransferases are potential cancer drug targets. Here, a protocol is presented to assess small molecules for DNA methyltransferase inhibition. This assay utilizes an endonuclease to couple DNA methylation to fluorescence generation and allows for enzyme activity to be monitored in real time.&#160;

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) ...

DetectSyn: A Rapid, Unbiased Fluorescent Method to Detect Changes in Synapse Density

Synapses are the site of communication between neurons. Neuronal circuit strength is related to synaptic density, and the breakdown of synapses is characteristic of disease states like major depressive disorder (MDD) and Alzheimer's disease. Traditional techniques to investigate synapse numbers include genetic expression of fluorescent markers (e.g., green fluorescent protein (GFP)), dyes that fill a neuron (e.g., carbocyanine dye, DiI), and immunofluorescent detection of spine markers (e.g., postsynaptic density 95 (PSD95)). A major caveat to these proxy techniques is that they only identify postsynaptic changes. Yet, a synapse is a connection between a presynaptic terminal and a postsynaptic spine. The gold standard for measuring synapse formation/elimination requires time-consuming electron microscopy or array tomography techniques. These techniques require specialized training and costly equipment. Further, only a limited number of neurons can be assessed and are used to represent changes to an entire brain region. DetectSyn is a rapid fluorescent technique that identifies changes to synapse formation or elimination due to a disease state or drug activity. DetectSyn utilizes a rapid proximity ligation assay to detect juxtaposed pre- and postsynaptic proteins and standard fluorescent microscopy, a technique readily available to most laboratories. Fluorescent detection of the resulting puncta allows for quick and unbiased analysis of experiments. DetectSyn provides more representative results than electron microscopy because larger areas can be analyzed than a limited number of fluorescent neurons. Moreover, DetectSyn works for in vitro cultured neurons and fixed tissue slices. Finally, a method is provided to analyze the data collected from this technique. Overall, DetectSyn offers a procedure for detecting relative changes in synapse density across treatments or disease states and is more accessible than traditional techniques.

DetectSyn is an unbiased, rapid fluorescent assay that measures changes in relative synapse (pre- and postsynaptic engagement) number across treatments or disease states. This technique utilizes a proximity ligation technique that can be used both in cultured neurons and fixed tissue.