Arginyltransferase ATE1 Catalyzes Midchain Arginylation of Proteins at Side Chain Carboxylates In Vivo

Wang, Junling; Han, Xuemei; Wong, Catherine C. L.; Cheng, Hong; Aslanian, Aaron; Xu, Tao; Leavis, Paul C.; Röder, Heinrich; Hedstrom, Lizbeth; Yates, John R.; Kashina, Anna

doi:10.1016/j.chembiol.2013.12.017

Cited by 78 publications

(92 citation statements)

References 19 publications

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“…Although the a 1 ion of a dimethylated arginine was not detected in the corresponding spectra, we exclude the possibility that the identified arginylation may represent a sidechain arginylation for several reasons. First, although sidechain arginylation was detected in mouse applying the same experimental setup (29), there is to our knowledge no report on sidechain arginylation that was preceded by deamidation of a glutamine or asparagine residue into a glutamic acid or an aspartic acid residue, respectively. Second, the modified Gln 30 was the most N-terminal amino acid identified across all measurements suggesting that it was the apparent N-terminus of PpATAD3.1.…”

Section: Resultsmentioning

confidence: 99%

“…In contrast, arginylation of glutamic acid residues can also target proteins via p62 (Sequestosome-1) binding to autophagy-mediated degradation in mammalian cell lines (27). Further, ATE-mediated arginylation can also occur on acidic side-chains of non-N-terminal amino acids (28,29). Although ATE activity was demonstrated quite early in plants (30), the functional investigation of protein arginylation in plants started only recently.…”

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confidence: 99%

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Identification of Targets and Interaction Partners of Arginyl-tRNA Protein Transferase in the Moss Physcomitrella patens

Hoernstein

Mueller

Fiedler

et al. 2016

Molecular & Cellular Proteomics

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Protein arginylation is a posttranslational modification of both N-terminal amino acids of proteins and sidechain carboxylates and can be crucial for viability and physiology in higher eukaryotes. The lack of arginylation causes severe developmental defects in moss, affects the low oxygen response in Arabidopsis thaliana and is embryo lethal in Drosophila and in mice. Although several studies investigated impact and function of the responsible enzyme, the arginyltRNA protein transferase (ATE) in plants, identification of arginylated proteins by mass spectrometry was not hitherto achieved. In the present study, we report the identification of targets and interaction partners of ATE in the model plant Physcomitrella patens by mass spectrometry, employing two different immuno-affinity strategies and a recently established transgenic ATE:GUS reporter line (Schuessele et al., 2016 New Phytol., DOI: 10.1111/nph.13656). Here we use a commercially available antibody against the fused reporter protein (␤-glucuronidase) to pull down ATE and its interacting proteins and validate its in vivo interaction with a class I small heatshock protein via Fö rster resonance energy transfer (FRET). Additionally, we apply and modify a method that already successfully identified arginylated proteins from mouse proteomes by using custom-made antibodies specific for N-terminal arginine. As a result, we identify four arginylated proteins from Physcomitrella patens with high confidence.

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Identification of Targets and Interaction Partners of Arginyl-tRNA Protein Transferase in the Moss Physcomitrella patens

Hoernstein

Mueller

Fiedler

et al. 2016

Molecular & Cellular Proteomics

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“…The same group described evidence that some isoforms of R-transferase were specific for substrates bearing N-terminal Cys (93). Other recent publications by the same laboratory suggested, on the basis of MS analyses, that the Ate1 R-transferase is capable of arginylating not only ␣-amino groups of specific N-terminal residues but also ␥-carboxyl groups of internal (non-N-terminal) Asp or Glu (94,95). This suggestion followed a report, by another group, that described a modified 13-residue neurotensin hormone that A yellow oval denotes the rest of a protein substrate.…”

mentioning

confidence: 88%

“…These compartments are distinct from the currently known locations of the cytosolic/ nuclear Ate1 R-transferase (96). A subsequent publication suggested that Ate1 is capable of arginylating not only the ␣-amino groups of N-terminal residues but also ␥-carboxyl groups of internal (non-N-terminal) Asp and Glu in natural proteins and described several proteins, including actin, dystrophin, myosin, titin, and tubulin, that appeared to contain, according to MSbased data, internal Asp or Glu residues conjugated to Arg (94).…”

Section: Use Of Celluspots Assays To Address Reports About Arginylatimentioning

confidence: 99%

“…B, rectangle on the right, the otherwise identical assay was carried out in the presence of purified mouse Ate1 1B7A R-transferase. Note that "negative" peptide spots, containing 11-residue peptides bearing non-canonical (non-Asp, non-Glu, and non-Cys) N-terminal residues, exhibited negligible (undetectable) levels of [ 14 (87,94,96). As a part of initial quality controls, and also to verify that peptides on CelluSpots arrays could actually serve as efficacious substrates of purified mouse Ate1 R-transferase isoforms, the arginylation reaction was optimized on slides containing duplicate copies of a peptide array, in either the presence or the absence of a purified mouse Ate1 R-transferase isoform ( Fig.…”

Section: Use Of Celluspotsmentioning

confidence: 99%

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Analyzing N-terminal Arginylation through the Use of Peptide Arrays and Degradation Assays

Wadas

Piatkov

Brower

et al. 2016

Journal of Biological Chemistry

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N␣ -terminal arginylation (Nt-arginylation) of proteins is mediated by the Ate1 arginyltransferase (R-transferase), a component of the Arg/N-end rule pathway. This proteolytic system recognizes proteins containing N-terminal degradation signals called N-degrons, polyubiquitylates these proteins, and thereby causes their degradation by the proteasome. The definitively identified ("canonical") residues that are Nt-arginylated by R-transferase are N-terminal Asp, Glu, and (oxidized) Cys. Over the last decade, several publications have suggested (i) that Ate1 can also arginylate non-canonical N-terminal residues; (ii) that Ate1 is capable of arginylating not only ␣-amino groups of N-terminal residues but also ␥-carboxyl groups of internal (non-N-terminal) Asp and Glu; and (iii) that some isoforms of Ate1 are specific for substrates bearing N-terminal Cys residues. In the present study, we employed arrays of immobilized 11-residue peptides and pulse-chase assays to examine the substrate specificity of mouse R-transferase. We show that amino acid sequences immediately downstream of a substrate's canonical (Nt-arginylatable) N-terminal residue, particularly a residue at position 2, can affect the rate of Nt-arginylation by R-transferase and thereby the rate of degradation of a substrate protein. We also show that the four major isoforms of mouse R-transferase have similar Nt-arginylation specificities in vitro, contrary to the claim about the specificity of some Ate1 isoforms for N-terminal Cys. In addition, we found no evidence for a significant activity of the Ate1 R-transferase toward previously invoked non-canonical N-terminal or internal amino acid residues. Together, our results raise technical concerns about earlier studies that invoked non-canonical arginylation specificities of Ate1.The N-end rule pathway is a set of intracellular proteolytic systems whose unifying feature is the ability to recognize and polyubiquitylate proteins containing N-terminal (Nt) 2 degradation signals called N-degrons, thereby causing the processive degradation of these proteins by the proteasome (Fig. 1, A and B) (1-13). Recognition components of the N-end rule pathway are called N-recognins. In eukaryotes, N-recognins are E3 ubiquitin (Ub) ligases that can target N-degrons. Some N-recognins contain several substrate-binding sites and thereby can recognize (bind to) not only N-degrons but also specific internal (non-N-terminal) degradation signals (Fig. 1A) (14 -18). The main determinant of a protein's N-degron is either an unmodified or chemically modified N-terminal residue. Another determinant of an N-degron is an internal Lys residue(s). It functions as a site of protein polyubiquitylation, is often engaged stochastically (in competition with other "eligible" lysines), and tends to be located in a conformationally disordered region (2,9,19,20). Bacteria also contain the N-end rule pathway, but Ub-independent versions of it (21-26).Regulated degradation of proteins and their natural fragments by the N-end rule pathway has been s...

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Ablation of arginyl‐tRNA‐protein transferase in oligodendrocytes impairs central nervous system myelination

Palandri

Farías

et al. 2021

Glia

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Addition of arginine (Arg) from tRNA can cause major alterations of structure and function of protein substrates. This post-translational modification, termed protein arginylation, is mediated by the enzyme arginyl-tRNA-protein transferase 1 (Ate1).Arginylation plays essential roles in a variety of cellular processes, including cell migration, apoptosis, and cytoskeletal organization. Ate1 is associated with neuronal functions such as neurogenesis and neurite growth. However, the role of Ate1 in glial development, including oligodendrocyte (OL) differentiation and myelination processes in the central nervous system, is poorly understood. The present study revealed a peak in Ate1 protein expression during myelination process in primary cultured OLs. Post-transcriptional downregulation of Ate1 reduced the number of OL processes, and branching complexity, in vitro. We conditionally ablated Ate1 from OLs in mice using 2 0 ,3 0 -cyclic nucleotide 3 0 -phosphodiesterase-Cre promoter ("Ate1-KO" mice), to assess the role of Ate1 in OL function and axonal myelination in vivo. Immunostaining for OL differentiation markers revealed a notable reduction of mature OLs in corpus callosum of 14-day-old Ate1-KO, but no changes in spinal cord, in comparison with wild-type controls. Local proliferation of OL precursor cells was elevated in corpus callosum of 21-day-old Ate1-KO, but was unchanged in spinal cord. Five-month-old Ate1-KO displayed reductions of mature OL number and myelin thickness, with alterations of motor behaviors. Our findings, taken together, demonstrate that Ate1 helps maintain proper OL differentiation and myelination in corpus callosum in vivo, and that protein arginylation plays an essential role in developmental myelination.

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Arginyltransferase ATE1 Catalyzes Midchain Arginylation of Proteins at Side Chain Carboxylates In Vivo

Cited by 78 publications

References 19 publications

Identification of Targets and Interaction Partners of Arginyl-tRNA Protein Transferase in the Moss Physcomitrella patens

Identification of Targets and Interaction Partners of Arginyl-tRNA Protein Transferase in the Moss Physcomitrella patens

Analyzing N-terminal Arginylation through the Use of Peptide Arrays and Degradation Assays

Ablation of arginyl‐tRNA‐protein transferase in oligodendrocytes impairs central nervous system myelination

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