Biology:Arginylation

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Short description: Arginylation Post-translational modification
In arginylation, arginine (pictured above) is added to proteins.

Arginylation is a post-translational modification in which proteins are modified by the addition of arginine (Arg) at the N-terminal amino group or side chains of reactive amino acids by the enzyme, arginyltransferase (ATE1). Recent studies have also revealed that hundreds of proteins in vivo are arginylated, proteins which are essential for many biological pathways. While still poorly understood in a biological setting, the ATE1 enzyme is highly conserved which suggests that arginylation is an important biological post-translational modification.

Examples of ATE1 targets which have been identified include ornithine decarboxylase.,[1] thyroglobulin,[2] insulin,[3] and neurotensin.[4]

Discovery

In 1963, a group of researchers observed that specific radioactive amino acids were being incorporated into proteins obtained from ribosome-free cell and tissue extracts.[5] This incorporation of amino acids into ribosome-lacking cells was first observed in prokaryotes using leucine (Leu) and phenylalanine (Phe), and was further discovered in mammalian liver extracts using arginine. The incorporation of other amino acids into ribosome-lacking cells failed to yield similar results, suggesting that the mechanism was specific to leucine and phenylalanine in bacteria and arginine in mammals.[6] One of the most interesting aspects of arginylation is that the amino acids used for arginylation are transferred from aminoacyl tRNAs onto the target protein, without the use of any other translational components. This way of modifying proteins post-translationally does not occur in any other amino acid addition to proteins, such as in glycylation,[7] glutamylation,[8] and tyrosination[9][10] making arginylation truly unique.

Upon discovery of this modification and its mechanism, further research was performed to identify an enzyme or enzymes which promote this modification. After identifying the enzyme responsible for this modification in both plants[11] and guinea-pig hair follicles,[12] it was cloned and characterized in yeast and given the name ATE1[13] due to its ability. Later studies have also identified various genes which code for ATE1 enzymes in multiple species, leading to the conclusion that ATE1 is present in all eukaryotes.[11][13][14]

Target sites

N-terminus

General structure of the N-terminus of an amino acid within a peptide (in arginylation, this amino acid is commonly asparagine or glutamine).

Upon the identification of the early targets of arginylation by ATE1 (in vitro and in vivo), a pattern emerged. This pattern showed that ATE1 displayed a high affinity for proteins and peptides containing the acidic amino acids asparagine or glutamine which were exposed on the N-terminal side of the protein or peptide. Further studies aided by high precision mass spectrometry have revealed hundreds of proteins from different cells and tissues which have been arginylated.[15][16] Several of these proteins also displayed arginylation at their N-chain termini, but contained residues other than asparagine or glutamine.[5] As such, arginylation studies are still in the introductory stages and further research into the specificity of arginylation must be performed.

However, the assumption that arginylation only occurs at the N-terminus severely limited the amount of proteins which were likely to be arginylated. This is due to the fact that, if the preference of arginylation to occur only at the N-terminus assumption was true, then arginylation would never be able to happen on intact proteins due to protein sequences beginning with methionine at the N-terminus and not the preferred asparagine or glutamine. This assumption was soon proved false when a protein was discovered with an arginylated residue in the middle of its sequence.

Mid-chain

Although N-terminus arginylation was originally thought to be the only site for targeting by ATE1 enzymes, it has recently been discovered that arginylation may also occur in the middle of the peptide chain of a protein. The first discovery of this unprecedented modification occurred when neurotensin, a biological peptide found in the central nervous system, was isolated from cells and it was discovered that arginine was attached to a mid-chain glutamine residue.[4] This discovery changed the view of how arginylation occurs, as this meant that there may be ways to modify and arginylate intact proteins.

In an effort to determine the prevalence of mid-chain arginylation, a mass spectrometry screen of various peptides was performed. The results from this experiment revealed a plethora of various proteins which contained modified asparagine and glutamine residues present in the middle of their peptide chain, and further studies determined that ATE1 could also be mediating this reaction. Indeed, this discovery changed the biological scope of arginylation by suggesting that arginylation can also occur on fully intact proteins, not just on the N-terminus of protein fragments or pre-processed proteins.[5]

Consequences

In 1986, the N-end rule was elucidated, and it states that the identity of the amino acid at the N-terminus of the protein's amino acid sequence determines the half-life of the protein. In an effort to determine the effects of arginylation on the half-life of proteins, several studies were performed using modified yeast proteins. These studies revealed that when proteins were engineered to include N-termini which had been arginylated, the modified proteins were metabolically unstable.[17][18][19] Furthermore, it was also discovered that protein ubiquitination and degradation become more likely to occur when a protein is arginylated.[20] The evidence gathered from these experiments make it clear that arginylation in vivo leads to the degradation of proteins with asparagine and glutamine residues at their N-termini.

However, there have also been several recent studies which have shown that protein degradation may not be the prevalent function of arginylation, but that this modification may also be important for certain proteins to function correctly. For instance, when arginylation occurs on beta amyloid proteins, the proteins are guided into their proper alpha helical shape and are also prevented from misfolding and aggregating.[21] Another protein which benefits from arginylation is calreticulin because when modified, its role during endoplasmic reticulum stress is facilitated, rather than it being removed from cells entirely.[22][23] As both degradation and facilitation effects of arginylation have been identified and studied, it is clear that arginylation has an important role in protein regulation within cells.

Regulation

Due to it being a lesser understood post-translational modification, arginylation and its regulation in vivo still remains largely esoteric. The expression of ATE1 can vary significantly within different tissues, but its levels within these tissues peak at mid-development[24] but begin to decline as an organism ages.[5][25] It has also been observed that a variety of physiological compounds and drugs are able to affect the incorporation of arginine in vivo, but it is hypothesized that this occurs in a non-specific manner.[26] As such, it has been theorized that inhibitors and activators which regulate ATE1 activity, and therefore arginylation, may exist in vivo.

Arginylation's ability to make proteins metabolically unstable, as observed in yeast, makes proteins which have been modified in this way an attractive target for removal. One of the well characterized arginylation regulators is the ubiquitin dependent protein degradation which quickly degrades and removes harmful proteins. This important regulator of arginylation facilitates the specificity of this post-translational modification and efficiently removes proteins which were not meant to be arginylated in vivo.[27]

Lastly, an unproven but highly attractive mechanism of regulating arginylation in vivo suggests the use of de-arginylation enzymes which may be able to remove an arginine that has been added post-translationally to proteins. Enzymes such as Aminopeptidase B and Carboxypeptidase B are able to remove arginine from a proteins N-terminus and from side chain carboxyl groups, respectively, but do not specifically target arginylated sites. The proposed de-arginylation enzymes are theorized to act in the same way as the previously mentioned enzymes Aminopeptidase B and Carboxypeptidase B, but would differ in the fact that they specifically target arginylated protein substrates. Although these enzymes have not been discovered as of yet, the search for and discovery of these enzymes is an exciting path for further studies.

Pathways regulated by

File:Arginylation-Dependent-Neural-Crest-Cell-Migration-Is-Essential-for-Mouse-Development-pgen.1000878.s016.ogv Initially written off as a non-essential process due to the ATE1 knockout in yeast, later studies have shown arginylation plays a significant role in several biological processes. The knockout of ATE1 in mice and Drosophila resulted in embryonic lethality for both species. Further studies using the mouse model to observe the effects of ATE1 knockout in the development of the organism revealed that the gene loss resulted in abnormal cardiac and craniofacial morphogenesis, impaired angiogenesis, and the ability of cells to undergo meiosis. Postnatally, ATE1 knockout resulted in weight loss, infertility, and mental retardation. Additionally, observing the effects of ATE1 deletion in Arabidopsis thaliana, a model plant organism, revealed defective shoot and leaf development, abnormal seed germination, and delayed leaf senescence. The dysfunctions resulting from the knockout of the ATE1 enzyme therefore suggest that arginylation is necessary for many physiological pathways within eukaryotes.

See also

References

  1. ↑ "Post-translational arginylation of ornithine decarboxylase from rat hepatocytes". The Biochemical Journal 267 (2): 343–348. April 1990. doi:10.1042/bj2670343. PMID 2334397. 
  2. ↑ "Enzymatic modification of proteins. 4. Arginylation of bovine thyroglobulin". The Journal of Biological Chemistry 246 (5): 1481–1484. March 1971. doi:10.1016/s0021-9258(19)76997-x. PMID 5101774. 
  3. ↑ "Evidence that oxidized proteins are substrates for N-terminal arginylation". Neurochemical Research 23 (11): 1411–1420. November 1998. doi:10.1023/A:1020706924509. PMID 9814552. 
  4. ↑ 4.0 4.1 "A novel form of neurotensin post-translationally modified by arginylation". The Journal of Biological Chemistry 280 (42): 35089–35097. October 2005. doi:10.1074/jbc.m502567200. PMID 16087676. 
  5. ↑ 5.0 5.1 5.2 5.3 "Protein Arginylation: Over 50 Years of Discovery". Protein Arginylation. Methods in Molecular Biology. 1337. New York, NY: Springer New York. 2015. pp. 1–11. doi:10.1007/978-1-4939-2935-1_1. ISBN 978-1-4939-2934-4. 
  6. ↑ "Protein arginylation, a global biological regulator that targets actin cytoskeleton and the muscle". Anatomical Record 297 (9): 1630–1636. September 2014. doi:10.1002/ar.22969. PMID 25125176. 
  7. ↑ "Polyglycylation of tubulin: a posttranslational modification in axonemal microtubules". Science 266 (5191): 1688–1691. December 1994. doi:10.1126/science.7992051. PMID 7992051. Bibcode: 1994Sci...266.1688R. 
  8. ↑ "Glutamylated tubulin: diversity of expression and distribution of isoforms". Cell Motility and the Cytoskeleton 55 (1): 14–25. May 2003. doi:10.1002/cm.10107. PMID 12673595. 
  9. ↑ "Incorporation of L-tyrosine, L-phenylalanine and L-3,4-dihydroxyphenylalanine as single units into rat brain tubulin". European Journal of Biochemistry 59 (1): 145–149. November 1975. doi:10.1111/j.1432-1033.1975.tb02435.x. PMID 1204603. 
  10. ↑ "Release of tyrosine from tyrosinated tubulin. Some common factors that affect this process and the assembly of tubulin". FEBS Letters 73 (2): 147–150. February 1977. doi:10.1016/0014-5793(77)80968-x. PMID 838053. 
  11. ↑ 11.0 11.1 "An arginyl-transfer ribonucleic Acid protein transferase from cereal embryos". Plant Physiology 52 (1): 13–16. July 1973. doi:10.1104/pp.52.1.13. PMID 16658490. 
  12. ↑ "Arginine transferase activity in homogenates from guinea-pig hair follicles". The Journal of Investigative Dermatology 67 (5): 582–586. November 1976. doi:10.1111/1523-1747.ep12541685. PMID 977987. 
  13. ↑ 13.0 13.1 "Cloning and functional analysis of the arginyl-tRNA-protein transferase gene ATE1 of Saccharomyces cerevisiae". The Journal of Biological Chemistry 265 (13): 7464–7471. May 1990. doi:10.1016/s0021-9258(19)39136-7. PMID 2185248. 
  14. ↑ "Identification of mammalian arginyltransferases that modify a specific subset of protein substrates". Proceedings of the National Academy of Sciences of the United States of America 102 (29): 10123–10128. July 2005. doi:10.1073/pnas.0504500102. PMID 16002466. Bibcode: 2005PNAS..10210123R. 
  15. ↑ "Global analysis of posttranslational protein arginylation". PLOS Biology (Public Library of Science) 5 (10): e258. October 2007. doi:10.1371/journal.pbio.0050258. OCLC 679480183. PMID 17896865. 
  16. ↑ "Identification of N-terminally arginylated proteins and peptides by mass spectrometry". Nature Protocols 4 (3): 325–332. 2009-02-19. doi:10.1038/nprot.2008.248. PMID 19229197. 
  17. ↑ "In vivo half-life of a protein is a function of its amino-terminal residue". Science 234 (4773): 179–186. October 1986. doi:10.1126/science.3018930. PMID 3018930. Bibcode: 1986Sci...234..179B. 
  18. ↑ "Universality and Structure of the N-end Rule". Journal of Biological Chemistry 264 (28): 16700–16712. 1989. doi:10.1016/s0021-9258(19)84762-2. PMID 2506181. https://authors.library.caltech.edu/107818/1/J.%20Biol.%20Chem.-1989-Gonda-16700-12.pdf. 
  19. ↑ "The N-end rule". Cold Spring Harbor Symposia on Quantitative Biology 60: 461–478. 1995-01-01. doi:10.1101/SQB.1995.060.01.051. PMID 8824420. 
  20. ↑ "Post-translational addition of an arginine moiety to acidic NH2 termini of proteins is required for their recognition by ubiquitin-protein ligase". The Journal of Biological Chemistry 265 (26): 15511–15517. September 1990. doi:10.1016/s0021-9258(18)55426-0. PMID 2168415. 
  21. ↑ "The post-translational incorporation of arginine into a beta-amyloid peptide increases the probability of alpha-helix formation". NeuroReport 7 (1): 326–328. December 1995. doi:10.1097/00001756-199512290-00078. PMID 8742481. 
  22. ↑ "Calreticulin-dimerization induced by post-translational arginylation is critical for stress granules scaffolding". The International Journal of Biochemistry & Cell Biology 45 (7): 1223–1235. July 2013. doi:10.1016/j.biocel.2013.03.017. PMID 23567256. 
  23. ↑ "Arginylated calreticulin at plasma membrane increases susceptibility of cells to apoptosis". The Journal of Biological Chemistry 287 (26): 22043–22054. June 2012. doi:10.1074/jbc.m111.338335. PMID 22577148. 
  24. ↑ "Alternative splicing results in differential expression, activity, and localization of the two forms of arginyl-tRNA-protein transferase, a component of the N-end rule pathway". Molecular and Cellular Biology 19 (1): 182–193. January 1999. doi:10.1128/mcb.19.1.182. PMID 9858543. 
  25. ↑ "Arginyl-tRNA transferase activity as a marker of cellular aging in peripheral rat tissues". Experimental Gerontology 15 (1): 53–64. 1980. doi:10.1016/0531-5565(80)90023-6. PMID 7409020. 
  26. ↑ "Posttranslational arginylation as a global biological regulator". Developmental Biology 358 (1): 1–8. October 2011. doi:10.1016/j.ydbio.2011.06.043. PMID 21784066. 
  27. ↑ "Arginylation-dependent regulation of a proteolytic product of talin is essential for cell-cell adhesion". The Journal of Cell Biology 197 (6): 819–836. June 2012. doi:10.1083/jcb.201112129. PMID 22665520. 



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