Using this method, which concurrently assesses Asp4DNS, 4DNS, and ArgAsp4DNS (eluting in sequence), a precise evaluation of arginyltransferase activity is possible, alongside identification of unfavorable enzymes within the 105000 g supernatant of tissue samples.
Arginylation assays, which involve chemically synthesized peptide arrays on cellulose membranes, are outlined in this description. This assay allows for a simultaneous comparison of arginylation activity across hundreds of peptide substrates, enabling analysis of arginyltransferase ATE1's specificity towards its target site(s) and the surrounding amino acid sequence. For the successful dissection of the arginylation consensus site and the forecasting of arginylated proteins within eukaryotic genomes, this assay was previously utilized in studies.
We describe a biochemical assay utilizing a microplate format for evaluating ATE1-catalyzed arginylation. The assay can be used for high-throughput screens to identify small molecule inhibitors and activators of ATE1, extensive analysis of AE1 substrate interactions, and similar research endeavors. Initially, we employed this screen on a collection of 3280 compounds, pinpointing two that demonstrably impacted ATE1-regulated processes both within and outside of living cells. The assay relies on in vitro arginylation of beta-actin's N-terminal peptide by ATE1, but its scope extends to encompass other substrates acted upon by ATE1.
Herein is described a standard in vitro arginyltransferase assay employing bacterially-expressed and purified ATE1 in a minimal component system consisting of Arg, tRNA, Arg-tRNA synthetase, and the arginylation substrate. Using crude ATE1 preparations extracted from cells and tissues, assays of this type were pioneered in the 1980s; these assays were subsequently optimized for use with bacterially expressed recombinant protein. By employing this assay, ATE1 activity can be measured in a simple and effective manner.
The preparation of pre-charged Arg-tRNA, utilizable in arginylation reactions, is detailed in this chapter. While arginyl-tRNA synthetase (RARS) usually participates in the process of arginylation by continuously charging tRNA with arginine, it might be essential to separate the charging and arginylation processes to independently manipulate reaction conditions and carry out analyses such as kinetic measurements and assessing the effects of diverse substances. The arginylation process can be facilitated by pre-charging tRNAArg with Arg and then separating it from the RARS enzyme in such cases.
This method offers a fast and efficient means of obtaining a concentrated sample of the target tRNA, which is further modified post-transcriptionally by the intracellular machinery of the host cells, E. coli. This preparation, though containing a blend of all E. coli tRNA, yields the targeted enriched tRNA in high quantities (milligrams) with notable effectiveness for in vitro biochemical testing. Within our laboratory, arginylation is conducted routinely with this.
In vitro transcription is employed in this chapter to detail the preparation of tRNAArg. Using tRNA produced by this method, in vitro arginylation assays are greatly facilitated by aminoacylation with Arg-tRNA synthetase, either immediately during the reaction or as a preliminary step for creating a purified Arg-tRNAArg preparation. Other chapters within this book detail the process of tRNA charging.
We outline the steps involved in the expression and subsequent purification of recombinant ATE1, a protein derived from genetically modified E. coli strains. This method allows for a straightforward and convenient one-step isolation process for milligram amounts of soluble, enzymatically active ATE1, resulting in a purity of nearly 99%. We also delineate a protocol for the expression and purification of E. coli Arg-tRNA synthetase, indispensable for the arginylation assays detailed in the subsequent two chapters.
A simplified version of the method, as detailed in Chapter 9, is presented in this chapter for the convenient and speedy evaluation of intracellular arginylation activity in live cells. see more Transfection of a GFP-tagged N-terminal actin peptide into cells yields a reporter construct; this method aligns with the technique described in the preceding chapter. Reporter-expressing cells can be harvested and analyzed directly via Western blotting to evaluate arginylation activity. An arginylated-actin antibody, along with a GFP antibody as a reference, is essential for this analysis. Direct comparison of different reporter-expressing cell types is feasible in this assay, despite the unmeasurability of absolute arginylation activity, thereby allowing for an evaluation of the effects of genetic background or treatment. The method's elegance and diverse biological utility led us to present it as a unique and distinct protocol.
This description outlines an antibody technique for assessing the enzymatic action of arginyltransferase1 (Ate1). An assay procedure relies on the arginylation of a reporter protein containing the N-terminal peptide from beta-actin, a known endogenous target of Ate1, and a C-terminal GFP tag. The reporter protein's arginylation level is assessed via immunoblot, utilizing an antibody targeting the arginylated N-terminus, whereas the substrate's total quantity is determined using an anti-GFP antibody. This method allows for the convenient and accurate assessment of Ate1 activity present in yeast and mammalian cell extracts. This method successfully determines the impact of mutations on critical amino acids within Ate1, as well as the effects of stress and other contributing factors on its functional activity.
The N-end rule pathway, understood through research in the 1980s, illustrated the ubiquitination and degradation of proteins due to the addition of N-terminal arginine. Physio-biochemical traits Following ATE1-dependent arginylation, several test substrates are found to efficiently utilize this mechanism; however, its application is limited to proteins possessing additional N-degron features, including a ubiquitination-accessible lysine located nearby. By analyzing the degradation of arginylation-dependent substrates, researchers could ascertain ATE1 activity in cells indirectly. The substrate for this assay, frequently E. coli beta-galactosidase (beta-Gal), allows for straightforward measurement of its concentration using standardized colorimetric assays. A method for rapidly and effortlessly characterizing ATE1 activity in diverse species during the identification of arginyltransferases is presented here.
A protocol for in vivo study of protein arginylation is detailed, focusing on the measurement of 14C-Arg incorporation into proteins of cultured cells. The conditions specified for this unique modification address the biochemical needs of the ATE1 enzyme, and the modifications necessary to distinguish between post-translational protein arginylation and the de novo synthesis pathway. Representing an optimal method for recognizing and validating possible ATE1 substrates, these conditions apply to diverse cell lines or primary cultures.
Our early work in 1963, which identified arginylation, has spurred subsequent investigations aimed at determining how its activity impacts crucial biological processes. Across diverse experimental setups, we used cell- and tissue-based assays to determine the level of acceptor proteins and the activity of ATE1. These assays exhibited a notable link between arginylation and age-related changes, a discovery which may contribute to a more comprehensive understanding of the significance of ATE1 in the framework of normal biology and disease therapeutics. We present the original techniques for assessing ATE1 activity in tissues, correlating these results with pivotal biological stages.
Early research efforts in protein arginylation, performed before the advent of widespread recombinant protein expression, often relied upon the fractional separation of proteins present within native tissues. This procedure, developed by R. Soffer in 1970, was a response to the 1963 discovery of arginylation. In this chapter, the detailed procedure originally published by R. Soffer in 1970, derived from his article and refined by collaboration with R. Soffer, H. Kaji, and A. Kaji, is presented.
Transfer RNA's participation in post-translational protein modification using arginine has been demonstrated in vitro through studies of axoplasm extracted from the giant axons of squid, and further confirmed in injured and regenerating vertebrate nerve systems. The highest activity level in nerve and axoplasm is observed in a particular fraction of a 150,000g supernatant, which contains high molecular weight protein/RNA complexes but is devoid of any components measuring less than 5 kDa. Arginylation, and protein modification by other amino acids, is conspicuously missing from the more purified, reconstituted fractions. To ensure maximal physiological activity, the data emphasizes the importance of recovering reaction components from high molecular weight protein/RNA complexes. Modèles biomathématiques Injured and developing vertebrate nerves demonstrate a significantly greater arginylation level than intact nerves, indicating a possible function in nerve injury/repair and axonal outgrowth.
Biochemical research in the late 1960s and early 1970s revolutionized the understanding of arginylation, leading to the first determination of ATE1's function and its substrate specificity. A summary of the recollections and insights from the period of research, extending from the original arginylation discovery to the identification of the arginylation enzyme, is presented in this chapter.
Protein arginylation, identified in 1963 as a soluble activity within cell extracts, is the process that mediates the incorporation of amino acids into proteins. The near-accidental nature of this discovery did not hinder the research team's commitment; their persistence has forged a new frontier in research. The original identification of arginylation, and the initial methodologies for proving its presence within biological systems, are discussed in this chapter.