Deletion of mFICD AMPylase alters cytokine secretion and affects visual short-term learning in vivo

Abstract

Fic domain-containing AMP transferases (fic AMPylases) are conserved enzymes that catalyze the covalent transfer of AMP to proteins. This posttranslational modification regulates the function of several proteins, including the ER-resident chaperone Grp78/BiP. Here we introduce a mouse FICD (mFICD) AMPylase knockout mouse model to study fic AMPylase function in vertebrates. We find that mFICD deficiency is well tolerated in unstressed mice. We also show that mFICD-deficient mouse embryonic fibroblasts are depleted of AMPylated proteins. mFICD deletion alters protein synthesis and secretion in splenocytes, including that of IgM, an antibody secreted early during infections, and the proinflammatory cytokine IL-1β, without affecting the unfolded protein response. Finally, we demonstrate that visual nonspatial short-term learning is stronger in old mFICD−/− mice than in wild-type controls while other measures of cognition, memory, and learning are unaffected. Together, our results suggest a role for mFICD in adaptive immunity and neuronal plasticity in vivo. Fic domain-containing AMP transferases (fic AMPylases) are conserved enzymes that catalyze the covalent transfer of AMP to proteins. This posttranslational modification regulates the function of several proteins, including the ER-resident chaperone Grp78/BiP. Here we introduce a mouse FICD (mFICD) AMPylase knockout mouse model to study fic AMPylase function in vertebrates. We find that mFICD deficiency is well tolerated in unstressed mice. We also show that mFICD-deficient mouse embryonic fibroblasts are depleted of AMPylated proteins. mFICD deletion alters protein synthesis and secretion in splenocytes, including that of IgM, an antibody secreted early during infections, and the proinflammatory cytokine IL-1β, without affecting the unfolded protein response. Finally, we demonstrate that visual nonspatial short-term learning is stronger in old mFICD−/− mice than in wild-type controls while other measures of cognition, memory, and learning are unaffected. Together, our results suggest a role for mFICD in adaptive immunity and neuronal plasticity in vivo. The posttranslational regulation of protein function is a fundamental concept in biology. To manage protein activity, dedicated enzymes attach specific chemical modifications to individual proteins, the presence of which affects the behavior and activity of the modified proteins. These modifications, called posttranslational modifications (PTMs), govern essential biological processes. They are implicated in cancer, neurodegeneration, and cardiovascular diseases, among others. The covalent addition of an AMP moiety to the side chain of exposed threonine and serine residues has emerged as a new paradigm to control the activity of the essential ER-resident chaperone BiP. This process, AMPylation, is catalyzed by metazoan AMP transferases (AMPylases) that contain a filamentation induced by c-AMP (fic) domain. Fic domain-containing AMPylases (fic AMPylases) are highly conserved and are present in a single copy in most metazoans, including Caenorhabditis elegans (FIC-1), Drosophila melanogaster (dfic), Mus musculus (mFICD), and Homo sapiens (FICD) (1Woolery A.R. Luong P. Broberg C.A. Orth K. AMPylation: Something old is new again.Front. Microbiol. 2010; 1: 113Crossref PubMed Scopus (51) Google Scholar, 2Itzen A. Blankenfeldt W. Goody R.S. Adenylylation: Renaissance of a forgotten post-translational modification.Trends Biochem. Sci. 2011; 36: 221-228Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 3Truttmann M.C.P.H.L. rAMPing up stress signaling: Protein AMPylation in metazoans.Trends Cell Biol. 2017; 27: 608-620Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). Metazoan fic AMPylases are bifunctional: using a single active site, these enzymes catalyze both the transfer of AMP to surface-exposed threonine and serine hydroxyl groups and the removal of AMP groups from modified residues (deAMPylation) (4Casey A.K. Moehlman A.T. Zhang J. Servage K.A. Krämer H. Orth K. Fic-mediated deAMPylation is not dependent on homodimerization and rescues toxic AMPylation in flies.J. Biol. Chem. 2018; 293: 1550Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar, 5Moehlman A.T. Casey A.K. Servage K. Orth K. Krämer H. Adaptation to constant light requires Fic-mediated AMPylation of BiP to protect against reversible photoreceptor degeneration.Elife. 2018; 7e38752Crossref PubMed Scopus (12) Google Scholar, 6Preissler S. Rato C. Perera L.A. Saudek V. Ron D. FICD acts bifunctionally to AMPylate and de-AMPylate the endoplasmic reticulum chaperone BiP.Nat. Struct. Mol. Biol. 2017; 24: 23-29Crossref PubMed Scopus (45) Google Scholar, 7Preissler S. Rohland L. Yan Y. Chen R. Read R.J. Ron D. AMPylation targets the rate-limiting step of BiP's ATPase cycle for its functional inactivation.Elife. 2017; 6e29428Crossref PubMed Scopus (41) Google Scholar, 8Veyron S. Oliva G. Rolando M. Buchrieser C. Peyroche G. Cherfils J. A Ca2+-regulated deAMPylation switch in human and bacterial FIC proteins.Nat. Commun. 2019; 10: 1142Crossref PubMed Scopus (14) Google Scholar). The switch between AMPylation and deAMPylation is proposed to involve enzyme dimerization, the exchange of Mg2+ with Ca2+ ions in the active site, and the subsequent switch from an open to a closed conformation (4Casey A.K. Moehlman A.T. Zhang J. Servage K.A. Krämer H. Orth K. Fic-mediated deAMPylation is not dependent on homodimerization and rescues toxic AMPylation in flies.J. Biol. Chem. 2018; 293: 1550Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar, 5Moehlman A.T. Casey A.K. Servage K. Orth K. Krämer H. Adaptation to constant light requires Fic-mediated AMPylation of BiP to protect against reversible photoreceptor degeneration.Elife. 2018; 7e38752Crossref PubMed Scopus (12) Google Scholar, 6Preissler S. Rato C. Perera L.A. Saudek V. Ron D. FICD acts bifunctionally to AMPylate and de-AMPylate the endoplasmic reticulum chaperone BiP.Nat. Struct. Mol. Biol. 2017; 24: 23-29Crossref PubMed Scopus (45) Google Scholar, 7Preissler S. Rohland L. Yan Y. Chen R. Read R.J. Ron D. AMPylation targets the rate-limiting step of BiP's ATPase cycle for its functional inactivation.Elife. 2017; 6e29428Crossref PubMed Scopus (41) Google Scholar, 8Veyron S. Oliva G. Rolando M. Buchrieser C. Peyroche G. Cherfils J. A Ca2+-regulated deAMPylation switch in human and bacterial FIC proteins.Nat. Commun. 2019; 10: 1142Crossref PubMed Scopus (14) Google Scholar, 9Perera L.A. Rato C. Yan Y. Neidhardt L. McLaughlin S.H. Read R.J. Preissler S. Ron D. An oligomeric state-dependent switch in the ER enzyme FICD regulates AMPylation and deAMPylation of BiP.EMBO J. 2019; 38e102177Crossref PubMed Scopus (17) Google Scholar). The latter is stabilized by interactions between an inhibitory glutamate and a nearby arginine residue, which aligns an inhibitory α-helix such that the catalytic core preferentially binds AMP over ATP, catalyzing deAMPylation (6Preissler S. Rato C. Perera L.A. Saudek V. Ron D. FICD acts bifunctionally to AMPylate and de-AMPylate the endoplasmic reticulum chaperone BiP.Nat. Struct. Mol. Biol. 2017; 24: 23-29Crossref PubMed Scopus (45) Google Scholar). If the interactions between these residues are prevented or resolved, fic AMPylases adopt an open conformation that favors Mg2+ and ATP recruitment to the active site, enabling AMPylation of target proteins (10Engel P. Goepfert A. Stanger F.V. Harms A. Schmidt A. Schirmer T. Dehio C. Adenylylation control by intra- or intermolecular active-site obstruction in Fic proteins.Nature. 2012; 482: 107-110Crossref PubMed Scopus (108) Google Scholar). Thus, replacing the critical inhibitory glutamate residue with a glycine (FICD(E234G)) converts the enzyme to a constitutively active AMPylase (10Engel P. Goepfert A. Stanger F.V. Harms A. Schmidt A. Schirmer T. Dehio C. Adenylylation control by intra- or intermolecular active-site obstruction in Fic proteins.Nature. 2012; 482: 107-110Crossref PubMed Scopus (108) Google Scholar, 11Truttmann M.C. Cruz V.E. Guo X. Engert C. Schwartz T.U. Ploegh H.L. The Caenorhabditis elegans protein FIC-1 is an AMPylase that proteins, and PubMed Scopus Google Scholar, H. A.R. C. D. Krämer H. Orth K. protein BiP during endoplasmic reticulum Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). AMPylation of the ER-resident on chaperone in an and to the of proteins S. Rohland L. Yan Y. Chen R. Read R.J. Ron D. AMPylation targets the rate-limiting step of BiP's ATPase cycle for its functional inactivation.Elife. 2017; 6e29428Crossref PubMed Scopus (41) Google Scholar, S. Rato C. Chen R. R. S. Ron D. AMPylation BiP activity to protein in the endoplasmic PubMed Scopus Google Scholar). BiP ATP is and the of BiP is to with proteins (6Preissler S. Rato C. Perera L.A. Saudek V. Ron D. FICD acts bifunctionally to AMPylate and de-AMPylate the endoplasmic reticulum chaperone BiP.Nat. Struct. Mol. Biol. 2017; 24: 23-29Crossref PubMed Scopus (45) Google Scholar, L.A. Rato C. Yan Y. Neidhardt L. McLaughlin S.H. Read R.J. Preissler S. Ron D. An oligomeric state-dependent switch in FICD regulates AMPylation and deAMPylation of the chaperone BiP.EMBO J. 2019; PubMed Scopus Google Scholar). The of BiP AMPylation and (4Casey A.K. Moehlman A.T. Zhang J. Servage K.A. Krämer H. Orth K. Fic-mediated deAMPylation is not dependent on homodimerization and rescues toxic AMPylation in flies.J. Biol. Chem. 2018; 293: 1550Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar, H. A.R. C. D. Krämer H. Orth K. protein BiP during endoplasmic reticulum Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, A.K. Orth K. in AMPylation and 2018; PubMed Scopus Google or A C. J. D. S. and of the PubMed Scopus Google Scholar, A. Chen C.A. A. S. A between fic induced by and the unfolded protein Biol. Chem. Full Text Full Text PDF PubMed Scopus Google BiP addition to fic AMPylases also a of proteins M.C. modifications of the chaperone Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M.C. D. Ploegh H.L. AMPylation and of Sci. A. 2018; PubMed Scopus Google Scholar, M.C. X. L. M. J. D. Ploegh H.L. AMPylation targets and the Sci. A. 2017; PubMed Scopus Google Scholar, M.C. Ploegh H.L. rAMPing up stress signaling: Protein AMPylation in metazoans.Trends Cell Biol. 2017; 27: 608-620Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar, M.C. Cruz V.E. Guo X. Engert C. Schwartz T.U. Ploegh H.L. The Caenorhabditis elegans protein FIC-1 is an AMPylase that affects to PubMed Scopus Google Scholar, A. V. P. Orth K. P. AMPylation: A to the of protein Chem. Biol. PubMed Scopus (17) Google Scholar, P. T. K. S. A for of protein PubMed Scopus Google Scholar, P. M. S. FICD activity and AMPylation human Commun. PubMed Scopus Google Scholar, M. M. of AMPylation a chemical Cell Full Text Full Text PDF PubMed Scopus Google Scholar, M. J. C. C. V. H. C. A. of targets of Fic enzymes by covalent Chem. PubMed Scopus Google Scholar). fic AMPylases are also present in the and the M.C. Cruz V.E. Guo X. Engert C. Schwartz T.U. Ploegh H.L. The Caenorhabditis elegans protein FIC-1 is an AMPylase that proteins, and PubMed Scopus Google Scholar, M.C. X. L. M. J. D. Ploegh H.L. AMPylation targets and the Sci. A. 2017; PubMed Scopus Google Scholar, R. S. an for of Cell Sci. 2019; PubMed Scopus Google Scholar). in AMPylation and of constitutively active fic AMPylases is toxic and human A. Chen C.A. A. S. A between fic induced by and the unfolded protein Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M.C. S. J. Ploegh H.L. as to target Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, A.R. M. D. M. of the protein and for its Full Text Full Text PDF PubMed Scopus Google and M.C. X. L. M. J. D. Ploegh H.L. AMPylation targets and the Sci. A. 2017; PubMed Scopus Google as well as M.C. D. Ploegh H.L. AMPylation and of Sci. A. 2018; PubMed Scopus Google and (4Casey A.K. Moehlman A.T. Zhang J. Servage K.A. Krämer H. Orth K. Fic-mediated deAMPylation is not dependent on homodimerization and rescues toxic AMPylation in flies.J. Biol. Chem. 2018; 293: 1550Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar). fic AMPylase deficiency is well tolerated in unstressed human the of the unfolded protein stress A. Chen C.A. A. S. A between fic induced by and the unfolded protein Biol. Chem. Full Text Full Text PDF PubMed Scopus Google and neuronal P. M. S. FICD activity and AMPylation human Commun. PubMed Scopus Google Scholar). show to the presence of proteins in M.C. D. Ploegh H.L. AMPylation and of Sci. A. 2018; PubMed Scopus Google Scholar). the most in fic AMPylase knockout is in which show in visual and from by BiP A.T. Casey A.K. Servage K. Orth K. Krämer H. Adaptation to constant light requires Fic-mediated AMPylation of BiP to protect against reversible photoreceptor degeneration.Elife. 2018; 7e38752Crossref PubMed Scopus (12) Google Scholar, M. H. X. Y. Orth K. Krämer H. in Drosophila requires of Fic in 2012; PubMed Scopus Google Scholar). the role of fic AMPylases in our of these enzymes is Here we the and of an mFICD-deficient mouse mFICD−/− mice are and are not We show that mFICD deletion alters synthesis and Finally, we that mFICD is in nonspatial short-term in vivo. Together, our results a role for mFICD function in adaptive immunity and neuronal plasticity in vertebrates. To the role of protein AMPylation in we to both mFICD-deficient and constitutively active mouse using We an that targets a to the of the and a to introduce the The of in than or in the mFICD that in not a single the constitutively active using T. M.C. Ploegh H.L. the of with by of PubMed Scopus Google Scholar). These results suggest that embryonic of constitutively active in the of a wild-type mFICD we a mouse a deletion in This deletion a in a and To the mFICD−/− we control and mFICD−/− using the is a which a and functional for Y. J. of a mouse on and PubMed Scopus Google Scholar). We that mFICD−/− mice to control for by results in We in and between control and mFICD−/− Together, these results that mFICD deficiency is well tolerated by mice AMPylation in is by FICD and the A. S. Servage K.A. Zhang J. J. M. M. Orth K. R. K. AMPylation by an conserved 2018; Full Text Full Text PDF PubMed Scopus Google Scholar). To mFICD deficiency alters the we mFICD−/− and control mouse embryonic with AMPylation, a to a on the modified proteins. We AMPylated proteins with and AMPylated proteins by results from mFICD−/− and a we proteins that AMPylated in wild-type these proteins several FICD including BiP and and proteins protein in both wild-type and mFICD−/− that AMPylated proteins in protein and stress Together, these results that FICD is for the regulation of and other proteins. that and mFICD−/− mice are in and and role in the an in and R. A. L. R. M. and both involve activity of the unfolded protein Biol. Chem. Full Text Full Text PDF PubMed Scopus Google we by mFICD we the of and in the We between and mFICD−/− mice in the of and and in and or and present in the in the and and in the and in mFICD−/− mice. an essential as an protein and by is for antibody and We the of mFICD deficiency on protein from and mFICD−/− mice and with or to cytokine which by mFICD deletion secretion on secretion of We also a in between and mFICD−/− mice This is and which the in the and is secreted by a that of the chain in mFICD−/− while the of other proteins that and the ER To the of mFICD deletion on protein and we on of for BiP activity in the of and L. D. The role of chain protein in Full Text PDF PubMed Scopus Google Scholar). from the using and for to We by with for and in the of for to protein from and and by by mFICD−/− mice of both the chain and the in This by of both and and of from the for both and mFICD−/− that the of in the mFICD−/− not ER control for These the We not in the synthesis or of other proteins AMPylation of BiP control over the of active BiP present in the ER S. Rohland L. Yan Y. Chen R. Read R.J. Ron D. AMPylation targets the rate-limiting step of BiP's ATPase cycle for its functional inactivation.Elife. 2017; 6e29428Crossref PubMed Scopus (41) Google Scholar, H. A.R. C. D. Krämer H. Orth K. protein BiP during endoplasmic reticulum Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, S. Rato C. Chen R. R. S. Ron D. AMPylation BiP activity to protein in the endoplasmic PubMed Scopus Google Scholar, A. Chen C.A. A. S. A between fic induced by and the unfolded protein Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). To of such control affects the and stress of we the induced during we from and with in the of or both and also in the of as by or or in the of targets BiP and We by targets of the by We in the between and mFICD−/− for of the that are targets of the and The induced is of antibody and from a induced by an of proteins. To mFICD−/− to the latter of we with several chemical of the with the from we in of the or targets by D. the mFICD regulates reversible photoreceptor which is critical for visual and to constant light A.T. Casey A.K. Servage K. Orth K. Krämer H. Adaptation to constant light requires Fic-mediated AMPylation of BiP to protect against reversible photoreceptor degeneration.Elife. 2018; 7e38752Crossref PubMed Scopus (12) Google Scholar, M. H. X. Y. Orth K. Krämer H. in Drosophila requires of Fic in 2012; PubMed Scopus Google Scholar). We mFICD−/− mice show of visual of the we visual and the light which in control as well as mFICD−/− To these we in which mice a a that in mice with S. of and mouse using a Sci. PubMed Scopus Google Scholar). mFICD deficiency not the to the early or in A and These results that mFICD deficiency not in mice. FICD activity and neuronal in human P. M. S. FICD activity and AMPylation human Commun. PubMed Scopus Google Scholar). To mFICD−/− in cognition, and memory, we control and mFICD−/− and of in These are to in visual short-term and and by and and for and of learning and 1: PubMed Scopus Google Scholar). in visual nonspatial short-term learning wild-type and mFICD−/− of A and mFICD−/− mice a between and while the of wild-type mice not and This that visual nonspatial short-term learning is stronger in old mFICD−/− mice than in wild-type we learning and of mFICD−/− and wild-type mice and of using a control and mFICD−/− mice well and removal of the wild-type and mFICD−/− the most in the that the in the learning and between control and mFICD−/− that mFICD deficiency not in Together, these results that and are not by mFICD Finally, to for we the to a new in the We that both control and mFICD−/− mice and of to the new and the new of the well Protein AMPylation in is as a that regulates the function of the ER-resident chaperone BiP as well as that of other proteins A.K. Orth K. in AMPylation and 2018; PubMed Scopus Google Scholar, M.C. Ploegh H.L. rAMPing up stress signaling: Protein AMPylation in metazoans.Trends Cell Biol. 2017; 27: 608-620Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). the of and are well in and the of fic AMPylase deficiency on is Here we a functional mFICD knockout We the and of mFICD deletion and and mFICD−/− mice our and of mFICD−/− mice not or These are in with in human and C. elegans in for FIC-1 both of which that the of is well tolerated in unstressed and A.T. Casey A.K. Servage K. Orth K. Krämer H. Adaptation to constant light requires Fic-mediated AMPylation of BiP to protect against reversible photoreceptor degeneration.Elife. 2018; 7e38752Crossref PubMed Scopus (12) Google Scholar, 7Preissler S. Rohland L. Yan Y. Chen R. Read R.J. Ron D. AMPylation targets the rate-limiting step of BiP's ATPase cycle for its functional inactivation.Elife. 2017; 6e29428Crossref PubMed Scopus (41) Google Scholar, 11Truttmann M.C. Cruz V.E. Guo X. Engert C. Schwartz T.U. Ploegh H.L. The Caenorhabditis elegans protein FIC-1 is an AMPylase that proteins, and PubMed Scopus Google Scholar, H. A.R. C. D. Krämer H. Orth K. protein BiP during endoplasmic reticulum Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, A. Chen C.A. A. S. A between fic induced by and the unfolded protein Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). with in which from in visual and A.T. Casey A.K. Servage K. Orth K. Krämer H. Adaptation to constant light requires Fic-mediated AMPylation of BiP to protect against reversible photoreceptor degeneration.Elife. 2018; 7e38752Crossref PubMed Scopus (12) Google Scholar, M. H. X. Y. Orth K. Krämer H. in Drosophila requires of Fic in 2012; PubMed Scopus Google we not or in in mFICD−/− mice. for these the presence of to proteins AMPylated by mFICD−/− in mice in target protein between and is to the role of mFICD in visual in a mFICD deficiency unstressed of most AMPylated proteins. with we find that mFICD both ER-resident and proteins M.C. Cruz V.E. Guo X. Engert C. Schwartz T.U. Ploegh H.L. The Caenorhabditis elegans protein FIC-1 is an AMPylase that proteins, and PubMed Scopus Google Scholar, M.C. D. Ploegh H.L. AMPylation and of Sci. A. 2018; PubMed Scopus Google Scholar, M.C. X. L. M. J. D. Ploegh H.L. AMPylation targets and the Sci. A. 2017; PubMed Scopus Google Scholar, P. M. S. FICD activity and AMPylation human Commun. PubMed Scopus Google Scholar, M. M. of AMPylation a chemical Cell Full Text Full Text PDF PubMed Scopus Google Scholar, M. J. C. C. V. H. C. A. of targets of Fic enzymes by covalent Chem. PubMed Scopus Google Scholar, R. S. an for of Cell Sci. 2019; PubMed Scopus Google Scholar, M.C. S. J. Ploegh H.L. as to target Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M. H. X. Y. Orth K. Krämer H. in Drosophila requires of Fic in 2012; PubMed Scopus Google Scholar, A. S. A. A. A. R. D. P. J. S. is a target of for Mol. Biol. 2019; PubMed Scopus Google Scholar). The that to target to AMPylation of BiP by mFICD regulates the of active BiP to a to ER stress S. Rohland L. Yan Y. Chen R. Read R.J. Ron D. AMPylation targets the rate-limiting step of BiP's ATPase cycle for its functional inactivation.Elife. 2017; 6e29428Crossref PubMed Scopus (41) Google Scholar). We show that in of mFICD activity in of synthesis in the ER secretion of is for and mFICD−/− BiP binds to the of to and the chain a light chain is for of to a in light chain or to between the and BiP to BiP We that deletion of mFICD secretion of in the is by to the active which is from the secretion R. L. S. P. The acts as a for secretion in J. 2018; PubMed Scopus Google Scholar). both the and protein secretion W. of protein Biol. 2018; Full Text Full Text PDF PubMed Scopus (48) Google Scholar). is that AMPylation a role in the regulation of the bacterial has also implicated in secretion M. M. J. S. C. S. S. P. from is a surface-exposed human in to 2010; PubMed Scopus Google Scholar). mFICD−/− mice not show of a to in most than wild-type control a we also in visual nonspatial short-term learning in old mFICD−/− mice as with wild-type controls and We to the of BiP and the of other AMPylated proteins such as and in the of on and protein AMPylation in the presence of proteins or in to that protein the role of in neuronal and our that mFICD deficiency is tolerated in the of stress and and processes. that FICD is in the regulation of stress that the of critical in of mFICD specific as to on the of protein AMPylation in or stress and the role of mFICD in and protein to and by the on and the and as in T. M.C. Ploegh H.L. the of with by of PubMed Scopus Google Scholar). We a using an to the for the FICD which in deletion or with to during of AMPylated proteins using a chemical as X. A.R. Luong P. M. J. J. X. G. Orth K. J. of AMPylation on human protein Cell Full Text Full Text PDF PubMed Scopus Google Scholar, M. Luong P. Orth K. A chemical for protein Chem. 2011; PubMed Scopus Google Scholar). we of mFICD−/− and wild-type with and the for This mFICD to as We the with to a to the groups on AMPylated proteins. AMPylated proteins using for from and with The and a with a using and using an in in a The against with and A of the is in We and mFICD−/− mice in of control and mFICD−/− mouse a to the in of and of as Y. J. of a mouse on and PubMed Scopus Google Scholar, J. and functional of mouse a proposed for PubMed Scopus Google Scholar). or in with and Protein using Protein in with by and to which in in and with and to Cell chain and light against mouse or and in using in a with The on the of the with a visual for and by The A the to in the the mice to to a the and with a The of the the from of the in and visual during and the to the from the the and the to find the and on the for to that not find the to by the mouse learning on and learning on and with the from the the mice in the the that the and to and in of the the the the in a from the and learning and mice of from mice on a to for This mice to to the of the The mice are on the for a These with a of between to as We a from S. of and mouse using a Sci. 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