Article

Amino Acid Building Blocks for Efficient Fmoc Solid-Phase Synthesis of Peptides Adenylylated at Serine or Threonine

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Abstract

The first straightforward building block based (non-interassembly) synthesis of peptides containing adenylylated serine and threonine residues is described. Key features include final global acidolytic protective group removal as well as full compatibility with standard Fmoc solid-phase peptide synthesis (SPPS). The described Thr-AMP SPPS-building block has been employed in the synthesis of the Thr-adenylylated sequence of human GTPase CDC42 (Ac-SEYVP-T(AMP)-VFDNYGC-NH(2)). Further, we demonstrate proof-of-concept for the synthesis of an Ser-adenylylated peptide (Ac-GSGA-S(AMP)-AGSGC-NH(2)) from the corresponding adenylylated serine building block.

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... There are currently no generic methods that allow specific enrichment of AMP-modified proteins. Anti-AMP antibodies, click chemistry-based approaches using ATP analogues and fluorescent or isotope-labelled ATP analogues have been used for target identification [10][11][12][13] . However, these approaches have several limitations: anti-AMP antibodies show a strong bias towards certain epitopes, leading to incomplete representation of the substrate profile, and nucleotide analogue-based methods suffer from competition of high levels of endogenous ATP in the cell or cell lysates. ...
... In the linear-regression analysis, insulin-like growth factor II receptor/cation-independent mannose 6-phosphate receptor (IGF2R/CI-MPR) and calcyclin binding protein/SIAH interacting protein (CACYBP/SIP) were the most enriched proteins from the MBP and 3xFLAG pulldowns, respectively. Subsequently, we confirmed IbpA-mediated AMPylation on recombinant IGF2R domains 4-6 and 14-C terminus as well as CACYBP in vitro by western blot analysis with an anti-AMP antibody (Fig. 5c,d) 12,13 . Due to challenges in purification and highly complex MS data, the modification sites for IGF2R remain to be determined. ...
Article
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Various pathogenic bacteria use post-translational modifications to manipulate the central components of host cell functions. Many of the enzymes released by these bacteria belong to the large Fic family, which modify targets with nucleotide monophosphates. The lack of a generic method for identifying the cellular targets of Fic family enzymes hinders investigation of their role and the effect of the post-translational modification. Here, we establish an approach that uses reactive co-substrate-linked enzymes for proteome profiling. We combine synthetic thiol-reactive nucleotide derivatives with recombinantly produced Fic enzymes containing strategically placed cysteines in their active sites to yield reactive binary probes for covalent substrate capture. The binary complexes capture their targets from cell lysates and permit subsequent identification. Furthermore, we determined the structures of low-affinity ternary enzyme–nucleotide–substrate complexes by applying a covalent-linking strategy. This approach thus allows target identification of the Fic enzymes from both bacteria and eukarya. Many bacterial pathogens release effector enzymes belonging to the large Fic family, which modify host targets with nucleotide monophosphates. Now, recombinantly produced Fic enzymes have been equipped with synthetic thiol-reactive nucleotide derivatives to make covalent binary probes. The reaction of modified Fic enzymes with their targets permits covalent substrate capture and the structural determination of low-affinity ternary enzyme–nucleotide–substrate complexes.
... Adenylylated serine and threonine residues have also been generated using a strategy based on the unprotected phosphodiesters with the allyl protective group of the phosphodiester linkage being cleaved at the end of the building block synthesis. 51 A similar building block strategy relied on the unprotected N6 nitrogen of the adenine. Due to the low nucleophilicity of the adenine NH 2 relative to the α-amino group of amino acids, the lack of protection is claimed to be compatible with standard peptide synthesis methods. ...
... The availability of amino acid building blocks for incorporation of both adenylylated Ser, Thr, and Tyr residues in peptides have simplified the production of specific antibodies. 50,51 In this respect, experiments for enrichment of PTM-modified proteins from cell lysates could be performed after general proteolytic digest of the cell lysates to both increase the accessibility of the PTMs and decrease nonspecific binding of the antibodies to random proteins in the lysate. 61 The recently derived antibodies have high affinity to Tyradenylylated proteins, with weak Thr-AMP-binding. ...
Article
In the cell, proteins are frequently modified covalently at specific amino acids with post-translational modifications, leading to a diversification of protein functions and activities. Since the introduction of high-resolution mass spectrometry, new posttranslational modifications are constantly being discovered. One particular modification is the adenylylation of mammalian proteins. In adenylylation, adenosine triphosphate (ATP) is utilized to attach an adenosine monophosphate at protein threonine or tyrosine residues via a phosphodiester linkage. Adenylylation is particularly interesting in the context of infections by bacterial pathogens during which mammalian proteins are manipulated through AMP attachment via secreted bacterial factors. In this review, we summarize the role and regulation of enzymatic adenylylation and the mechanisms of catalysis. We also refer to recent methods for the detection of adenylylated proteins by modification-specific antibodies, ATP analogues equipped with chemical handles, and mass spectrometry approaches. Additionally, we review screening approaches for inhibiting adenylylation and briefly discuss related modifications such as phosphocholination and phosphorylation.
... To further extend the Fmoc building-block approach to peptides adenylylated at serine and threonine, C.H. and co-workers developed a strategy that relied on the corresponding unprotected phosphodiesters. [29] Here, the allyl protective group of the phosphodiester linkage was removed in the last synthesis step, thereby making the phosphodiester monoanionic prior to activation and peptide coupling, and thus avoiding b-elimination. By employing this strategy, a number of peptides were prepared, for example the sequence of Thr-adenylylated human small GTPase Cdc42 (Scheme 2 D). ...
... The availability of amino acid building blocks for incorporation of both phosphocholinated and adenylylated Ser, Thr, and Tyr residues in peptides greatly simpli-fies the production of specific antibodies against these PTMs. [3,29,31] In this respect, experiments for enrichment of PTM-modified proteins from cell lysates can be performed after general proteolytic digestion of cell lysate, both to increase the accessibility of the PTMs and to decrease nonspecific binding of the antibodies to random proteins in the lysate. [34] Recently, the groups of C.H. (anti-Tyr-AMP) and Orth (anti-Thr-AMP) independently demonstrated the production of specific antibodies against adenylylated proteins and peptides. ...
Article
Editing the translations: Adenylylation and phosphocholination have recently been found as important post-translational modifications used by pathogenic bacteria during the infection process. This review discusses the combined use of chemical handles and specific antibodies for the identification of previously unknown substrates of these post-translational modifications in infected host cells.
... 8,9 We have demonstrated that peptides adenylylated at tyrosine, threonine, and serine could effectively and conveniently be synthesized relying on preformed adenylylated amino acid building blocks for Fmoc solid-phase peptide synthesis (SPPS). 10,11 Phosphocholination has only been reported on serine and threonine residues; however, we included tyrosine as well in our investigation, since phosphocholinated tyrosine is equally conceivable and might well be of biological relevance. The outlined building blocks ( Figure 2) consist of the N-terminal Fmoc protected amino acids onto which the choline moiety is attached via phosphodiesters in monoanionic form. ...
... Thus, the use of the unprotected phosphodiester has been shown to suppress the formation of the β-elimination product, since the monoanionic form provides a much poorer leaving group. 11 The phosphoimidate chemistry requires non-nucleophilic solvents, posing a challenge for the introduction of the cationic choline moiety, since the physicochemical properties of most choline salts allow only very limited solubility in suitable organic solvents. Schulz and co-workers reported the use of choline tosylate for the synthesis of phosphatidylcholine derivates according to the phosphoimidate protocol; 12 however, the tosylate interferes with the reactive phosphorus(III) species upon activation, thereby significantly lowering the yields. ...
Article
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