| 1. |
- Gulen, Burak, et al.
(författare)
-
Identification of targets of AMPylating Fic enzymes by co-substrate-mediated covalent capture
- 2020
-
Ingår i: Nature Chemistry. - : Nature Publishing Group. - 1755-4330 .- 1755-4349. ; 12:8, s. 732-739
-
Tidskriftsartikel (refereegranskat)abstract
- 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.
|
|
| 2. |
- Hoepfner, Dorothea, et al.
(författare)
-
Monoclonal Anti-AMP Antibodies Are Sensitive and Valuable Tools for Detecting Patterns of AMPylation
- 2020
-
Ingår i: iScience. - Cambridge : Cell Press. - 2589-0042. ; 23:12
-
Tidskriftsartikel (refereegranskat)abstract
- AMPylation is a post-translational modification that modifies amino acid side chains with adenosine monophosphate (AMP). Recently, a role of AMPylation as a universal regulatory mechanism in infection and cellular homeostasis has emerged, driving the demand for universal tools to study this modification. Here, we describe three monoclonal anti-AMP antibodies (mAbs) from mouse that are capable of protein backbone-independent recognition of AMPylation, in denatured (western blot) as well as native (ELISA, IP) applications, thereby outperforming previously reported tools. These antibodies are highly sensitive and specific for AMP modifications, highlighting their potential as tools for new target identification, as well as for validation of known targets. Interestingly, applying the anti-AMP mAbs to various cancer cell lines reveals a previously undescribed broad and diverse AMPylation pattern. In conclusion, these anti-AMP mABs will further advance the current understanding of AMPylation and the spectrum of modified targets.
|
|
